Biochemical and pharmacological characterizations of...
Transcript of Biochemical and pharmacological characterizations of...
Biochemical and pharmacological characterizations of the novel endogenous opioid peptide motifs and synthetic
nociceptin hexapeptide sequences
PhD Thesis
by
Engin Bojnik
Supervisors
Prof. Dr. Borsodi Anna
Prof. Dr. Benyhe Sándor
Institiute of Biochemistry
Biological Research Center of the Hungarian Academy of Sciences
Szeged, 2009
TABLE OF CONTENTS
ACKNOWLEDGEMENTS .................................................................................... i
ABBREVIATIONS................................................................................................ iii
I. INTRODUCTION ...................................................................................... 1
1. Historical overview of poppy plant and opium................................... 1
2. Opioid and nociceptin system............................................................ 2
3. Effector mechanisms ......................................................................... 4
4. Compounds targeting opioid receptors............................................... 5
5. Endogenous opioid peptides .............................................................. 6
6. Precursor polypeptides ...................................................................... 7
7. Pharmacology of the opioid and nociceptin system............................ 8
8. Opioid system in lower vertebrates.................................................. 10
8.1 Precursor polypeptides and opioid sequences .................................. 11
8.2 Nociception..................................................................................... 12
II. AIM OF THE STUDIES........................................................................... 13
III. MATERIALS AND METHODS .............................................................. 15
1. Radiochemicals ............................................................................... 15
2. Opioid and NOP receptor ligands .................................................... 15
3. Other chemicals............................................................................... 16
4. Animals........................................................................................... 16
5. Rat brain membrane preparations .................................................... 17
6. Preparation of hNOP-CHO cells ...................................................... 17
7. Receptor binding assays .................................................................. 18
8. [35S]GTPS binding assays .............................................................. 19
9. Isolated tissue bioassay.................................................................... 19
10. Calcium-ion mobilization experiments ............................................ 20
11. Internalization of the fluorescent receptor........................................ 21
12. Data analysis and terminology......................................................... 21
IV. RESULTS.................................................................................................. 23
NATURALLY OCCURRING NOVEL OPIOID PEPTIDES ................... 23
1. Bioinformatic examination of the opioid motifs in PDYN and PENK
precursor polypeptides of various species ........................................ 23
2. Receptor binding assays .................................................................. 26
3. [35S]GTPγS binding experiments ..................................................... 32
IN-VITRO PHARMACOLOGICAL CHARACTERIZATION OF NOVEL
NOP RECEPTOR SELECTIVE HEXAPEPTIDE, Ac-RYYRIR-ol......... 34
1. Receptor binding assays................................................................... 34
2. [35S]GTPγS binding assays .............................................................. 35
3. Electrically stimulated isolated tissues and mouse colon bioassay.... 36
4. Calcium ion mobilization experiments ............................................. 38
5. Internalization of the NOP receptors ................................................ 38
SELECTIVE AND HIGH AFFINITY LABELING OF NOP RECEPTORS
WITH THE HEXAPEPTIDE RADIOPROBE [3H]Ac-RYYRIK-ol ........ 40
Heterologous competition experiments with [3H]Ac-RYYRIK-ol ........ 40
V. DISCUSSION............................................................................................ 43
1. Identification of the Novel Naturally Occurring Opioid Peptides ..... 43
2. Pharmacological Characterization of the Ac-RYYRIR-ol ................ 47
3. Binding Characterization of the [3H]Ac-RYYRIK-ol ...................... 50
VI. SUMMARY............................................................................................... 52
VII. REFERENCES ......................................................................................... 54
VIII. LIST OF THESIS RELATED PUBLICATIONS.................................... 61
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ACKNOWLEDGEMENTS
I am sincerely thankful to my supervisor Dr. Borsodi Anna for her great guidance
support and for giving me the opportunity to study in her laboratory where I
completed my PhD studies. I also would like to thank her for involving me with
fruitful scientific collaborations.
I am indebted to my co-supervisor Dr. Benyhe Sándor for his excellent assistance,
scientific instructions, teachings, and his patience during my studies. I appreciate his
tolerance, calmness and truthfully scientist character with immeasurable knowledge.
I am very thankful to Dr. Girolamo Calo’ (Ferrara, Italy) for hosting me in his lab
were I could achieve the necessary pharmacological results for this thesis. I am also
thankful to Dr. Calo’s co-workers Dr. Carmella Fischetti and Dr. Valeria Camarda.
I am grateful to Dr. Magyar Anna and her student Babos Fruzsina (Budapest,
Hungary) for their help with synthesizing the majority of the peptides used in this
work.
I am thankful to Dr. Tóth Géza, Dr. Csaba Tömböly (Szeged, Hungary) and all of
their co-workers for the peptide synthesis and providing the radioligands necessary for
this thesis.
I would like to thank Dr. Ayfer Yalç nı (Izmir, Turkey), Dr. Maithe Corbani
(Montpellier, France), Dr. Raquel Rodriguez and to Dr. Ezequiel Marron (Salamanca,
Spain) for accepting me in their laboratories for short term visits and teaching me new
scientific techniques.
I am also very thankful to Dr Fekete Éva, Dr Farkas Tamás, Dr. Bagyánszki Mária
and Dr. Bakota Lídia for their help during my education.
I gratefuly thank to Hungarian Academy of Sciences, Biological Research Center,
Institute of Biochemistry for providing me 4 year PhD fellowship.
Many many thanks to all of my former and present colleagues in Opioid Research
Group, our technicians, and all of my friends whom I met during my stay in Szeged.
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Finally, my special thanks to Boynik Family (my mother Sebahat; father Hamit; sister
Elmas and brother Sezgin and his upcoming daughter) and to Valeria Biscotti for their
endless love and emotional supports, which was the greatest encouragement for my
studies.
At last I convey my respect to the entire scientific community especially for those
which are trying to survive in the difficult conditions and I would like to dedicate this
work to the Srebrenica victims.
Engin BOJNIK
Szeged, August 2009
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ABBRAVIATIONS
Ac Acetyl BSA Bovine serum albumin cAMP Adenosine-3,5’-cyclic monophosphate cDNA Complementary DNA copied from an mRNA coding for a protein;
it is inserted into surrogate host cells to cause them to express the protein
CHO Chinese hamster ovary CTAP D-Phe-Cys-Thr-D-Trp-Arg-Thr-Pen-Thr-NH2 DAMGO Tyr-D-Ala-Gly-nMe-Phe-Gly-ol DIDI Tyr-D-Ala-Phe-Glu-Ile-Ile-Gly-NH2 DOP delta-opioid peptide receptor DPDPE Tyr-D-Pen-Gly-Phe-D-Pen EC50 Potency - the agonist molar concentration that produces 50% of
the maximal possible effect of that agonist. EDTA Ethylenediamine-tetraacetic acid EGTA Ethylene glycol tetraacetic acid EKC Ethylketocyclazocine EL Exracellular loop Emax Efficacy - the maximal effect that an agonist can elicit in a given
tissue/preparation GDP Guanosin diphosphate G-protein Guanine nucleotide binding protein GPCR G-protein coupled receptor GPI Guinea pig ileum GTP Guanosin 5’-triphosphate GTPS Guanosine-5’-O-(3-thiotriphosphate) [35S]GTPS Guanosine-5’-[γ-35S]thiophosphate IL Intracellular loop IUPHAR International Union of Pharmacology J-113397 (±)trans-1-[1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-
ethyl-1,3- dihydro-2H-benzimidazol-2-one Kd Equilibrium dissociation constant of a radioligand-receptor
complex Ki equilibrium inhibition constant KOP kappa-opioid peptide receptor MOP mu-opioid peptide receptor mRNA Messenger ribonucleic acid mVD Mouse vas deferens N/OFQ Nociceptin/Orphanin FQ (Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-
Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln) NOP receptor Nociceptin/Orphanin FQ peptide receptor ORL-1 receptor Opioid receptor-like 1 pA2 Antagonist potency - the negative logarithm to the base 10 of the
antagonist molar concentration that makes it necessary to double the agonist concentration to elicit the original submaximal response.
PBS Phosphate-buffered saline PDYN Pro-dynorphin
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pEC50 Agonist potency - the negative logarithm to base 10 of the agonist molar concentration that produces 50% of the maximal possible effect of that agonist.
PEI Polyethyleneimine PENK Pro-enkephalin pKB Antagonist potency - when one single concentration of antagonist
is used. pKi Antilogarithm of the equilibrium inhibition constant PMSF Phenylmethylsulfonyl fluoride PNOC Pro-nociceptin POMC Pro-opiomelanocortin Ro-65-6570 (8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza spiro [4,5] decan-4-one) SEM Standard error of the mean TM Transmembrane Tris Tris-(hydroxymethyl)-aminomethane U-50488 2-(3,4-dichlorophenyl)-N-methyl-N-[(1R,2R)-2-pyrrolidin-1-
ylcyclohexyl]acetamide U-69593 (+)-(5α,7α,8β)-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro-[4,5]
dec-8-yl] UFP-101 [Nphe1,Arg14,Lys15]N/OFQ-NH2 UFP-102 [(pF)Phe4,Arg14,Lys15]N/OFQ-NH2 ZP-120 Ac-Arg-Tyr-Tyr-Arg-Trp-Lys-Lys-Lys-Lys-Lys-Lys-Lys-NH2
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INTRODUCTION
1. Historical overview of poppy plant and opium
The milky fluid extracted from the Papaver somniferum plant's seed-capsule
(‘poppyhead’) is highly narcotic after drying. This product is referred as opium. The
word ‘opium’ derives from the Greek word for juice of a plant; after all, opium is
prepared from the juice of the poppy plant. The name of the plant means, the "sleep-
bringing poppy", referring to its narcotic properties.
The medical historic writings of Theophrastus (3rd century B.C.) were the first known
written source mentioning opium. The Egyptians cultivated opium thebaicum in
famous poppy fields around 1300 B.C. Opium was used as a poison to let people to
death without pain, but it was also employed in medicine. In the ancient time, poppy
plant has been applied for anaesthesia, analgesia and for treating diarrhoea as well as
sleeping disorders. During the Middle Ages, many of the uses of opium were
appreciated. In 1680, Sydenham wrote: “Among the cures which it has given to man
to relieve his sufferings, none is as efficacious as opium” (Wong, 2003)
Fig. 1. Papaver somniferum L., Opium poppy.
Opioids have been the basis of pain treatment for thousands of years, and they remain
so today. Opioids are the collective names for alkaloid compounds directly derived
from opium and for many synthetic derivatives that have been created which imitate
the effects of those drugs, as well as the endogenous peptides that interact with the
opioid receptors. The poppy plant itself produces at least 20 different alkaloids. The
most important ones are the analgesic and narcotic morphine, the antitussive codeine,
the smooth muscle relaxant papaverine and the convulsant thebaine. The pain
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relieving effect of opium and later of pure morphine has been successfully used
against severe pain. The term ‘opiate’ includes all natural and synthetic drugs with
morphine like action. The old term opiates is now more and more replaced by the
term opioids which applies to any substance, whether endogenous or synthetic,
peptidic or non-peptidic, that produces morphine-like effects through an action on
opioid receptors (Trescot et al., 2008).
2. Opioid and nociceptin system
Opioids, like many other bioactive compounds, are acting through specific cellular
elements called receptors. Consistent with the ‘receptor theory’ established by Paul
Ehrlich: ‘Corpora non agunt nisi fixata’ (‘substances do not act unless bound’),
opioid binding cellular structures are opioid receptors. The discovery of opioid
receptors in 1973 by demonstrating the existence of specific binding sites for opioid
radioligands in neural cell membranes turned out to be a breakthrough in biochemical
pharmacology (Terenius, 1973; Pert and Snyder, 1973; Simon et al., 1973).
Receptor theory suggested also that opioid receptors in physiological conditions are
activated by endogeneous molecules within the body. These natural compounds were
designated ‘endorphins’ by fusing the terms ‘endogenous’ and ‘morphine’. The
intensive search for endogenous opioids resulted in the discovery of two related
pentapeptides methionine (Met-) and leucine (Leu-) enkephalin in 1975 (Hughes et
al., 1975). Endogenous opioid peptides and their G-protein-coupled receptors are
located in the central nervous system and peripheral tissues. The opioid system has
been studied to determine the intrinsic mechanism of modulation of pain and many
other physiological processes and to develop uniquely effective pain-control
substances with minimal abuse potential and fewer side effects (Prüll et al., 2003).
Opioids produce analgesia by binding to opioid receptors both within and outside the
central nervous system.
Following the elegant biochemical presentation of opioid receptors (Terenius, 1973;
Pert and Snyder, 1973; Simon et al., 1973), the first evidence for their heterogeneity
was achieved in the mid seventies and based upon detailed and comprehensive
pharmacological analyses of various opioid drugs (Martin et al., 1976; Gilbert and
Martin, 1976). Receptor-binding and cloning studies confirmed the existence of three
main receptor types: mu- (µ), delta- (δ), kappa- (κ). A fourth member of the opioid
peptide receptor family, the nociceptin/orphanin FQ (N/OFQ) receptor, was cloned in
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1994, which does not interact with any of the classical opiate ligands (Meunier et al.,
1995), but it is part of the opioid family based on extensive sequence homology.
First, the N-terminus of N/OFQ, FGGF, is highly reminiscent of the canonical YGGF
of the opioid peptides. Furthermore, the N/OFQ precursor protein exhibits several
analogous structures as compared to the opioid precursors: the active peptides are
located in the C-terminal part and seven Cys residues are found conserved at the N-
terminus of pronociceptin, prodynorphin, and proenkephalin (Nothacker et al., 1996).
Then, the NOP receptor shares more than 60% identity with the three opioid receptors
(Bunzow et al., 1994; Mollereau et al., 1994).Altogether, these data support the view
that the receptors, as well as the neuropeptide precursors of both the opioid and the
OFQ/N systems, have evolved from common ancestral genes.
In 2000, the Committee on Receptor Nomenclature and Drug Classification of the
International Union of Pharmacology (NC-IUPHAR, http:/www.iuphar.org) adopted
the terms MOP, DOP, KOP and NOP to indicate mu-, delta-, kappa- opioid and
nociceptin/orphanin FQ (N/OFQ) peptide receptors, respectively.
Fig. 2 Opioid receptor structure. EL -Extracellular loop; TM - Transmembrane helix;
IL - Intracellular loop
Opioid receptors are cell surface glycoproteins and they are tightly integrated into the
cell membranes. The human MOP, DOP, KOP and NOP genes encodes a heptahelical
protein comprised of 400, 372, 380 and 370 amino acids respectively. Several splice
variants of MOP (formerly MOR-1) have been cloned (Cadet et al., 2004). Only one
DOP gene has been cloned to date. Pharmacological experiments in rodents indicate
the subdivision into δ1 and δ2 receptors. From the binding characteristics of the
prototypical ligand ethylketocyclazocine, evidence for the subdivisions in κ1, κ2 and
κ3 has been provided (Paternak et al., 2004). Splice variants have been found in the
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human and mouse NOP receptor (Xie et al., 1999). These receptor variants differ in
their distribution in the central nervous system and in the rate of internalization and
desensitization upon agonist exposure but have similar binding and coupling
properties. MOP, DOP, KOP and NOP genes are located on paralogous (gene from
the same homologous superfamily found in another part of the genome of a particular
taxon, paralogous are the result of a gene duplication, not a lineage splitting event)
regions of human chromosomes which is evidence for gene duplication for opioid
receptors (Fredriksson et al., 2003). Human MOP and DOP genes are mapped to
chromosome 6 and 1, respectively, while KOP and NOP genes map to chromosomes
8 and 20.
All of the cloned opioid receptor types belong to the Gi/Go-coupled superfamily of
receptors (GPCRs). Such receptors are composed of a single polypeptide chain. The
main feature of these proteins is the presence of seven membrane-spanning sequences
containing hydrophobic amino acid side-chains permitting the integration of the
macromolecule into the lipid bilayer of the cell membrane by hydrophobic lipid-
protein interactions (Corbett et al., 1993). Covalent modifications, e.g. palmitoylation
of the C-terminal, and composition of disulphide bridge(s) between the extracellular
loops can help to maintain the properly folded active receptor conformation. All of
the opioid receptor types possess the same general structure of an extracellular N-
terminal region containing N-glycosylation recognition sites, the seven
transmembrane domains taking part in the formation of the binding pocket, and
intracellular C-terminal tail structure with phosphorylation- and G-protein interacting
sites (Fig. 2.). The water soluble connecting portions of the polypeptide chain form
three extracellular (facing outside) and three intracellular (located whithin the
cytoplasma) loops (Devi et al., 2001).
3. Effector mechanisms
Upon agonist ligand binding (morphine, enkephalin, N/OFQ etc.), receptor proteins
become activated due to conformational changes that results in the transduction of the
information signal. The major downstream element of opioid signaling whithin the
membrane is the activation of the Gα subunit of regulatory G-proteins (Gi/Gq and Go
types mainly). Activation of any type of opioid receptor consecutively inhibits the
enzyme adenylate cyclase via inhibitory G-proteins, resulting in a fall in the
intracellular level of the key second-messenger molecule cAMP and diminishes action
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potential firing (Chevlen, 2003). Conversely, opioid addicts undergoing withdrawal
suffer elevated cAMP levels and enhanced protein kinase A activity, resulting in
increased neurotransmitter release (Corbett et al., 2006).
4. Compounds targeting opioid receptors
The MOP is the classical target for morphine and mediates the analgesic and addictive
affects of the drug. Therefore, in MOP-deficient mice morphine does not exhibit
analgesic and positive reinforcing properties (Matthes et al., 1996). Endomorphin-1
and 2 are two pentapeptides that show the highest selectivity for this receptor (Zadina
et al., 1997). Another selective agonist is the synthetic peptide DAMGO (D-Ala2,
nMe-Phe4, Gly5-ol) enkephalin) (Handa et al., 1981). The action of MOP agonists are
competitively blocked by the antagonist naloxone, which is not absolutely specific for
MOPs. The somatostatin analogue CTAP (D-Phe-Cys-Thr-D-Trp-Arg-Thr-Pen-Thr-
NH2) has been found to be a more selective MOP antagonist (Pelton et al., 1986).
The DOP receptor is the primary target for met- and leu-enkephalin which also
exhibits affinities for MOP and KOP receptors. DIDI (Ile5,6deltorphin-2) and DPDPE
(D-Pen2, D-Pen5) enkephalin (Mosberg et al., 1983) are selective agonists, and
naltrindole is a selective antagonist on DOP.
Table 1. Opioid receptors and their selective ligands
The KOP receptor is the natural target for prodynorphin-derived peptides, such as
dynorphin A, dynorphin B, α-neoendorphin etc. The prototypical, although non-
selective ligand is ethylketocyclazocine (EKC). U-69593 and U-50488 are selective
agonists and norbinaltorphimine is an irreversible selective antagonist (Takemori et
al., 1988).
None of the endogenous opioid peptides or the opiate drugs shows high affinity for
the NOP receptor. An endogenous ligand that binds to NOP with high affinity has
been identified and termed nociceptin FQ (Meunier et al, 1995) or orphanin FQ
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(Reinscheid et al., 1995), currently also referred as N/OFQ.. Pseudopeptide bond
containing UFP-102 (Carrà et al., 2005) and non-peptide Ro-656570 (Rizzi et al.,
2001) are one of the most potent NOP agonists. Recently, peptide UFP-101 (Calo et
al., 2005, Arduin et al., 2007) and non peptide J-113397 (Kawamoto et al., 1999), a
drug with potent and selective antagonist activity at NOP receptors, has been
characterized.
Hexapeptides Ac-RYYRIK-NH2, Ac-RYYRWK-NH2, Ac-RYYRWR-NH2 etc. with
high affinity and selectivity to the NOP receptor were identified from a combinatorial
peptide library containing about 52 millions of compounds and reported to have
partial agonist properties at the NOP receptors (Dooley et al., 1996; Dooley et al.,
1997). These structures have also become templates for other peptide analogues with
additional chemical modifications, such as ZP-120: Ac-RYYRWKKKKKKK-NH2
(Rizzi et al., 2002), Ac-Arg-D-Cha-Qaa-D-Arg-D-pClPhe-NH2 (van Cauwenberghe et
al., 2004) or Ac-RYYRIR-NH2 (Ambo et al., 2007).
5. Endogenous opioid peptides
Natural opioid peptides, defined as endogenous peptides with opioid-like
pharmacological effects, are derived from distinct precursor polypeptides and have a
characteristic anatomical distribution. Precursor polypeptides are encoded in the
genome and are synthesized by protein translation using an mRNA template with the
help of the ribosomal machinery (Costa et al., 1987). Each precursor is subjected to
complex enzymatic cleavages, transport and other posttranslational modifications
resulting in the accomplished active peptides. Many non-neuronal cells, including
endocrine- and exocrine glands, cells of the immune system and heart, produce opioid
peptides (Cabot, 2001). These peptides play a regulatory role in many different
physiological systems.
Endogenous opioid peptides were first found and identified chemically in porcine
brain and pituitary (hypophysis) extracts by Scottish researchers (Hughes at al.,
1975), they reported the existence of an endogenous morphine-like substance that
interacted with morphine (MOP) and enkephalin (DOP) receptors. Later, they purified
these substances to homogeneity and named them enkephalin (‘in the head’).
Enkephalins are closely related pentapeptides with the structure of Tyr-Gly-Gly-Phe-
Met/Leu (or in the one-letter code: YGGPM/L).
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β-endorphin, a endogenous opioid peptide derived from the Pro-opiomelanocortin
which was discovered from camel pituitary extracts (Li et al.,, 1976) is equiactive at
MOP and DOP receptors with much lower affinity for KOP receptors (Corbett et al.,
2006). Met- and Leu-enkephalin have high affinities for DOP receptors, ten-fold
lower affinities for MOP receptors and negligible affinity for KOP receptors. In
addition, two endogenous tetrapeptides have been isolated that exhibit a high
selectivity for MOP receptors and have been called endomorphin-1 and endomorphin-
2 with the general structure of Tyr-Pro-Trp/Phe-Phe-NH2 (Zadina et al., 1997).
Opioid peptides and their receptors have subsequently been studied through the use of
immunohistochemistry (Covenas et al., 2004), and have been found both at a central
and at a peripheral levels.
Table 2. Precursor proteins and endogenous opioid peptides. *Presently unknown.
6. Precursor polypeptides
In mammals the endogenous opioid peptides are derived from four precursors: pro-
opiomelanocortin (POMC), pro-enkephalin (PENK), pro-dynorphin (PDYN) and pro-
nociceptin/orphanin FQ (PNOC) (Table 2) (Nakanishi et al., 1979; Noda et al., 1982;
Kakidani et al., 1982).
The opioid peptide precursor genes are located in different chromosomes in the
species investigated. Genetic structures are closely related, all opioid precursor genes
have only one introne within a region encoding the N-terminus of the full-length
precursor protein (Nikoshkov et al., 2005). Each prepro-polypeptide precursor, as
product of the source gene, undergoes post-translational modifications (processing)
that result in multiple bioactive peptides. These peptides share (with the exception of
nociceptin) the common N-terminal sequence of Tyr-Gly-Gly-Phe-(Met or Leu),
which has been termed either ‘opioid motif’ or ‘message sequence’; this pattern is
followed by various C-terminal extensions yielding peptides ranging from 5 to 31
residues in length. Each precursor polypeptides have N-terminal signal peptid
sequence for transport and release, cysteine rich N-terminal region, and differing
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numbers of opioid core sequences encompassed by specific recognition sites for
enzymatic cleavage. Pairs of basic amino acids, such as Lys-Arg (KR) or Lys-Lys
(KK) or Arg-Arg (RR) surround enkephalin (opioid) units in the precursor. This,
highly protonated positively charged dipeptide repeats determine recognition sites for
the processing endopeptidase enzymes, e.g., prohormone convertase.
POMC is a multifunctional precursor giving rise to adrenocorticotropin (ACTH), α-,
γ- and β-melanocyte stimulating hormone (MSH) but apparently only one opioid
peptide, β-endorphin (Li et al., 1976). Besides the pentapeptides Met-enkephalin
(ME) and Leu-enkephalins (LE), proenkephalin is processed in the heptapeptide Met-
enkephalin-Arg6-Phe7 (MERF) and the octapeptide Met-enkephalin-Arg6-Gly7-Leu8
(MERGL) (Table 2). PDYN is processed into dynorphin A, dynorphin A (1–8),
dynorphin B, α-, β-neoendorphin (Goldstein et al., 1981). The bioactive fragments of
PDYN, particularly dynorphin A and dynorphin B, have high affinity for KOP
receptors but also have significant affinity for MOP and DOP receptors (Danielson et
al., 1999). Other important feature of the mammalian PDYN is the lack of any Met-
enkephalin sequence in the precursor. N/OFQ was discovered by the first successful
application of the ‘reverse pharmacology approach’, i.e., the use of an orphan receptor
to identify its endogenous ligand and its processed from its precursor polypeptide
PNOC (Meunier et al., 1995; Reinscheid et al., 1995).
The organization of the PENK gene and the three other opioid precursor genes,
PDYN, POMC, and PNOC, suggests that these four genes share a common
evolutionary origin. From a phylogenetic point of view, PENK is believed to be the
ancestral form, the present POMC, PNOC and especially PDYN forms appeared at
later stages of evolution (Danielson and Dores, 1999; Dores et al., 2002). To date,
complete PENK, PDYN, POMC and PNOC cDNA sequences have been obtained for
multiple species of mammals, amphibians and fish. Attempts to clone the genes for
the endomorphin-1 and endomorphin-2 corresponding precursor molecules have not
been successful.
7. Pharmacology of the opioid and nociceptin system
For decades of synthetic efforts in the opioid field has been the search for an
alternative to morphine, which would induce powerful analgesia without the attendant
effects of respiratory depression and, above all, liability for abuse. Due to their
relatively rapid enzymatic decomposition and the limited transport across the blood
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brain barrier the pharmacological effects of the enkephalins are generally less than
that of morphine, at least in systemic administration.
Figure 3. Effects of the opioids and N/OFQ on major organ systems. Clinical indications
are noted in bold.
In humans, morphine-like drugs produce analgesia, drowsiness, changes in mood, and
mental clouding. Opioids have long been used to treat acute pain (such as post-
operative pain). They have also been found to be effective in severe, chronic,
disabling pain of terminal conditions such as cancer, and degenerative conditions such
as rheumatoid arthritis (Figure 3). A significant feature of the analgesia is that it
occurs without loss of consciousness (McQuay, 1999). Opioids are classified as
central nervous system depressants, i.e., they are cellular inhibitors of the neuronal
activity (Chevley, 2003). Other synthetic opioids (e.g., fentanyl) are also effective as
an analgesic (McQuay, 1999) or act as cough suppressants (e.g., codeine) (Mogil and
Paternak et al., 2001). Semi-synthetic opioids also have valuable medical uses either
as analgesics (e.g., oxycodone) or antitussive compounds (e.g., hydrocodone)(Mogil
and Paternak et al., 2001). At higher doses, effects include increased sedation or
euphoria, impaired concentration, reduced respiration and blood pressure, contraction
of pupils, constipation (Hargreaves and Joris, 1993) and, in some cases, rapid and
irregular heart rate (Raehal et al., 2005). Tolerance for opiates, but typically not for
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endogenous opioid peptides, develops fairly rapidly, making higher doses necessary
to maintain the desired intensity of effects (Stein et al., 1993). Most synthetic opioid
narcotics and heroin, are quite addictive, and regular use of them may result in severe
physical dependence, a serious and life-threatening health problem.
The N/OFQ is involved in a wide range of physiological responses with effects noted
in the nervous system (central and peripheral), the cardiovascular system, the airways,
the gastrointestinal tract, the urogenital tract and the immune system (Lambert, 2008)
(Figure 3). The effects in the nervous system are complex and have received much
attention. It is generally accepted that spinal N/OFQ is antinociceptive with many
features that are common to the classical members of the opioid family (Zeilhofer et
al., 2003). Nevertheless, when given supraspinally, N/OFQ reverses the effects of
opioids (anti-opioid action) with a whole-animal response that manifests as
hyperalgesia (Zeilhofer et al., 2003). In the brain, N/OFQ is affects the response to
stress, anxiety and locomotion (Chiou et al., 2007). In the cardiovascular system
N/OFQ produces bradycardia and hypotension; this response is similar to that
produced by classical opioids and more specifically to morphine used in the clinic.
8. Opioid system in lower vertebrates
The opioid/orphanin gene family provides a model system for analyzing the genome
duplication events. Studies done with proenkephalin gene provided a support for the
organizational arrangement which has been conserved throughout the extensive
radiation of the vertebrates (Dores et al., 2002, Dores et al., 2004). It has been
proposed that the evolution of the opioid/orphanin precursor gene family resulted
from successive duplication of the proenkephalin gene (‘Proenkephalin hypotheses`).
These duplications gave rise first, to the proopiomelanocortin gene, then to the
pronociceptin gene and most recently to the prodynorphin gene (Khalap et al., 2005).
Among the Gnathostomes (animals that have developed the trait of a jaw), the
organizational plan for the PENK precursor is remarkably conserved. For example in
lineages as diverse as mammals, cartilaginous fish and bony fish there are usually
seven opioid sequences present in this precursor (Khalap et al., 2005). Yet, within
vertebrate lineages there can be distinct sets of pentapeptide opioids (YGGFM or
YGGFL). In the Sarcopterygii, the sixth opioid position in lungfishes and anuran
amphibian proenkephalin genes encodes a Met-enkephalin (YGGFM) sequence
(Lecaude et al., 2000). However, in mammalian proenkephalin there is a Leu-
11
enkephalin (YGGFL) sequence at this position. These data are consistent with the
conclusion that the transition from a Met-enkephalin sequence to a Leu-enkephalin
sequence at the sixth opioid position in tetrapod proenkephalins occurred in the
ancestral proto-reptiles (Roberts et al., 2007).
There were a number of radioligand binding studies in brain homogenates from non-
mammalian species including fish (Gonzalez-Nunez et al., 2006), reptiles (Xia et al.,
2001) and birds (Martin et al 1992). However, the largest effort came from the study
of opioid binding sites in amphibian brain membranes such as Rana esculenta
(Benyhe et al., 1990; Benyhe et al., 1994; Benyhe et al., 1999; Wolleman et al., 1999).
8.1. Precursor polypeptides and opioid sequences
African clawed frog, Xenopus laevis, PDYN precursor contains a novel opioid
pentapeptide, an N-terminally positioned Ile5-enkephalin (IE) pentapeptide motif
(Pattee et al., 2003); this oligopeptide unit is absent in all mammalian PDYNs, but it
is also found in some fish PDYNs, such as the ones cloned from eel (Anguilla
rostrata, Alrubaian et al., 2006) and Nile tilapia (Oreochromis niloticus, Alrubaian et
al., 2006). Another unique enkephalin structure has been deduced from a partial
cDNA sequence of the African lungfish (Protopterus annectens) prodynorhin (Dores
et al., 2000). African and Australian lungfish are well known as ‘living fossils’
among evolutionary biologists. Lungfish were dominant and widespread during the
Paleozoic era, and the earliest fossils belong to the Devonian period; they are the
closest living relatives of land Vertebrates (Joss et al., 2006; Lee et al., 2006).
Lungfish prodynorphin encodes for Phe5-enkephalin (YGGFF) that is the second
example of a different non-mammalian enkephalin structure.
In the case of PENK derived octapeptides, ubiquitous YGGFMRGL sequence is
present in 26 mammals including the human. Several other mammalian octapeptides
exist, e.g., YGGFMRGY (Xenopus laevis, Ornithorhyncus anatinus), YGGFMRSY
(rainforest opossum, Monodelphis domestica), YGGFMRAL (cat and dog),
YGGFMRSL (mouse), YGGFMRGV (European rabbit, Oryctolagus cuniculus and
American pika, Ochotona princeps). Among other vertebrate animals, including fish,
amphibians, reptiles and birds, variability in the structure of the octapeptide is present
within the PENK polypeptide. Mouse PENK contains YGGFMRSL which is unique
among mammals, but is also present in the Australian lungfish (Neoceratodus
forsteri) and African lungfish (Protopterus annectens) proenkephalins (Sollars et al.,
12
2000; Dores et al., 2000). The double substituted YGGFMNGF sequence is present
in a cartilaginous fish species (Komorowski et al., 2004 direct submission), the
bullhead shark (Heterodontus portusjacksoni). While YGGFMRGY sequence occurs
in the Xenopus laevis and Xenopus tropicalis (Martens and Herbert, 1994).
YGGFMRSV octapeptide is included in the chicken and florida gar proenkephalin
(Khalap et al., 2005).
Mutations of nucleotide bases in the coding regions of the DNA lead to changes in
amino acid sequence, thus explain the differences observed at the amino acid level of
certain neuropeptides in various animal species. The more distantly related two
species are, the more substitutions can be found in one and the same neuropeptide.
Difference in the amino acid sequence may cause difference in biological function,
but usually, the orthologous family members seem to produce the same effect
(Holmgren and Jensen, 2001). The region carrying the biological activity is generally
the most conserved segment in neuropeptides.
8.2. Nociception
There is a wealth of information regarding nociception in mammalian systems,
however, recently non-mammalian vertebrates, amphibian and avian models, have
been extensively studied (Gentle, 1992; Stewens, 2003).
Nociceptors (sensory receptors that react to potentially damaging stimuli, such as
pain) are associated with free nerve endings and are usually of two fibre types, small
myelinated A-delta fibres and smaller unmyelinated C fibres (Lynn, 1994). Yet in the
the oldest living ancestor of the fishes, the lamprey, Petromyzon marinus, has only
unmyelinated fibres, yet electrophysiological recordings indicated slowly adapting
receptors that responded to noxious heat, therefore, possibly nociceptive (Matthews
and Wickelgren, 1978). Initial studies of the analgesic or antinociceptive effects of
opioids in amphibians (Rana pipiens) were conducted using non-selective opioid
agonists, endogenous opioid peptides, and antagonists (Pezalla et al., 1984a).
Tolerance to the analgesic effects of daily morphine administration was documented
(Stevens et al., 1993) and stress-induced release of endogenous opioids produced
analgesia which was potentiated by enkephalinase inhibitors and blocked by
naltrexone (Pezalla et al., 1984b). Those studies showed that both exogenous opioid
agonists and endogenous opioid peptides could raise the nociceptive threshold in
amphibians by an action at opioid receptors.
13
AIM OF THE STUDIES
Opioids are the most effective analgesic drugs for acute and chronic pain. Opioid
system consists of mu- (MOP) delta- (DOP) kappa (KOP) nociceptin (NOP) opioid
receptors and endogenous opioid peptides (enkephalins, -endorphin, dynorphins and
nociceptin or N/OFQ) (Corbett et al 2006). All the endogenous opioid peptides are
derived from four precursor polypeptides named as proenkephalin (PENK),
prodynorphin (PDYN), proopiomelanocortin (POMC) and pronociceptin (PNOC).
While PENK is source for the two enkephalin pentapeptides (Met- and Leu-
enkephalin, YGGFM and YGGFL respectively), PDYN is source for dynorphins.
Beside the enkephalins, PENK is also precursor for the Met-enkephalin extended
heptapeptide, met-enkephalin-Arg6-Phe7 (YGGFMRF) and octapeptide, met-
enkephalin-arg6-gly7-leu8 (YGGFMRGL) sequences (Patey and Rossier, 1986). Our
analysis of upto 55 different PENK and 39 PDYN sequences available in major
protein databases showed that the pentapeptide and octapeptide units are variable
among the different species studied.
Artificial hexapeptides isolated from peptide combinatorial chemical libraries are
amongst the most selective ligands for the NOP receptor. One leading compound in
this series is Ac-RYYRIK-NH2, developed by Dooley (Dooley et al., 1996). Recently
a close derivative with a reduced C-terminus, Ac-RYYRIK-ol has been described by
our group as a partial agonist at the NOP receptors using various in vitro and in vivo
studies (Gündüz et al., 2006). Being a partial agonist with intrinsic antagonist
potency, this hexapeptide alcohol is capable of inhibiting competitively some effects
mediated by full agonists at the NOP receptor. A further analogue, Ac-RYYRIR-ol,
representing the Lys6Arg6 replacement of its parent compound Ac-RYYRIK-ol, has
been synthesized aiming even better potency.
Present studies were devoted to characterize naturally occurring opioid penta- and
octapeptides as well as the synthetic NOP receptor selective hexapeptide analogue
Ac-RYYRIR-ol using biochemical, functional and pharmacological approach. In-vitro
biochemical assays were performed in rat brain membranes. Receptor-type selectivity
patterns were determined in radioreceptor binding experiments, while G-protein
activating properties of the peptides were investigated in [35S]GTPγS functional tests.
14
Main objective of the present studies were;
To collect all available PENK and PDYN sequences and aligning them to
characterize variability of the opiod peptides within the precursors,
To chemically synthesize and to characterize the novel opioid sequences (Table 3)
using comparative biochemical methods such as receptor binding and G-protein
activation assays,
To investigate the pharmacological properties of Ac-RYYRIR-ol using various in
vitro assays, such as receptor binding, G-protein activation, mouse deferens, guinea
pig ileum, and mouse colon bioassays, and calcium mobilization experiments,
To study the effective radiolabeling and detailed receptor binding properties of the
newly developed radioligand [3H]Ac-RYYRIK-ol to native and recombinant NOP
receptors.
Table 3. All the novel sequences studied in this thesis which includes, two non-mammalian enkephalin, four octapeptides together with its oxidized forms, one modified and one radiolabelled hexapeptide which selectively acts towards the NOP receptors
15
MATERIALS AND METHODS
1. Radiochemicals
[3H]Naloxone (28 Ci/mmol), [3H]DAMGO ([D-Ala2,NMePhe4,Gly5-ol]enkephalin;
41 Ci/mmol), [3H]Tyr1,Ile5,6deltorphin-2 (48 Ci/mmol), (Nevin et al.,1994) [3H]D-
Ala3-dynorphin-[1-11]amide (40 Ci/mmol) (Lung et al., 1995), [3H]Ac-RYYRIK-ol
(94 Ci/mmol) (Bojnik et al., 2009b), [Phenylalanyl-3H]nociceptin-amide (25
Ci/mmol) (Wollemann et al., 2008) were radiolabeled in the Isotope Laboratory of
BRC, Szeged. [3H]U-69,593 (55 Ci/mmol) and unlabelled U-69,593H that is (+)-
(5α,7α,8β)-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro-[4,5]dec-8-yl] were purchased
from Amersham Biosciences and the Upjohn Co., respectively, [Leucyl-3H]nociceptin
(TRK1047; specific activity 162 Ci/mmol were purchased from Amersham, U.K.
Guanosine-5-[-35S]-triphosphate (1204 Ci/mmol) was purchased from the Isotope
Institute Ltd. (Budapest, Hungary).
2. Opioid and NOP receptor ligands
All peptides used in these studies were chemically synthesized and not
extracted/purified from biological sources. Mammalian Leu- and Met-enkephalin
(YGGFL/M) and methionine extended octapeptide (YGGFMRGL) and N/OFQ used
in these studies were purchased from Bachem Feinbiochimica, Bubendorf,
Switzerland. Lungfish Phe-enkephalin (YGGFF),; non mammalian octapeptides,
YGGFMRGY (African clawed frog), YGGFMRSV (Chicken), YGGFMNGF (shark)
and YGGFMRSL (African lungfish) and NOP receptor selective hexapeptide
alcohols, Ac-RYYRIR-ol and Ac-RYYRIK-ol and other analogues (Kocsis et al.,
2004) were prepared at the Research Group for Peptide Chemistry of the Hungarian
Academy of Sciences, Budapest, Hungary, by liquid phase peptide synthesis. Frog Ile-
enkephalin (YGGFI) was prepared by automated solid phase peptide synthesis at the
Isotope Laboratory of BRC, Szeged, Hungary. Naloxone hydrochloride was kindly
provided by Du Pont de Nemours and buprenorphine was obtained from ICN-
Alkaloida Inc., (Tiszavasvari, Hungary). The NOP receptor antagonists
[Nphe1,Arg14,Lys15]nociceptin-NH2 (UFP-101) and (1-[(3R,4R)-1-cyclooctylmethyl-
3-hydroxy-methyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one; J-
113397) were kindly provided by Dr. Girolamo Calo' (Ferrara, Italy). All the
16
compounds used in these studies were purified by analytical HPLC methods, and the
structures were confirmed by mass spectrometry.
3. Other chemicals
MgCl2, KCl, EDTA, polyethylenimine (PEI), Tris(hydroxymethyl)aminomethane
(Tris), protease-free bovine serum albumin (protease-free BSA, fraction V),
guanosine 5’-diphosphate (GDP), guanosine-5’-O-(3-thiotriphosphate), GTPS were
purchased from Sigma-Aldrich (St. Louis, MO, USA). Morphine was obtained from
ICN Alkaloida, Tiszavasvári, Hungary. All the other chemicals used in this study
were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
4. Animals
Inbred Wistar rats (250–300 g body weight) and guinea pigs (R9strain) were housed
in the local animal house of the Biological Research Center (BRC, Szeged, Hungary).
Rats were kept in groups of four, allowed free access to standard food and tap water
and maintained on a 12-h light/dark cycle until the time of sacrifice. Male Swiss mice
(25–30 g), guinea pigs (300–350 g), and Sprague Dawley rats (300–350 g) were used
throughout the in vitro isolated tissue assays of Ac-RYYRIR-ol (Morini, Reggio
Emilia, Italy). Mice were housed in 425 x 266 x 17 155-mm cages (Techniplast,
Milan, Italy), eight animals/cage, under standard conditions (22ºC, 55 % humidity,
12-h light/dark cycle, light on at 7:00 am) with food (MIL, standard diet) and water ad
libitium for at least 3 days before experiments began. Animals were handled
according to the European Communities Council Directives (86/609/ECC) and the
Hungarian Act for the Protection of Animals in Research (XXVIII.tv. 32.§) and
Italian national regulations (D.L. 116/92). Accordingly, the number of animals and
their suffering were minimized.
5. Rat brain membrane preparations
Crude membrane fractions were prepared from Wistar rat brains according to the
method of Pasternak with small modification (Simon et al., 1986). Rats were
decapitated and the brains without cerebellum were quickly removed and washed
several times to remove any unwanted blood or tissue particles with chilled 50 mM
Tris–HCl (pH 7.4) buffer. The brains were blotted dry, weighed and suspended in five
17
volumes/weight of the original brain tissue with ice-cold 50 mM Tris–HCl buffer. The
brains were than homogenized at 1000 rpm with an electrically driven Braun Teflon-
glass rota-homogenizer at 4 °C, using 10–15 strokes of the homogenizer. The final
volume of the homogenate was made up to 30 volumes/weight of the brain and
filtered through four layers of gauze to remove any larger aggregates. After
centrifugation with a Sorvall RC5C centrifuge at 4000xg (18,000 rpm) for 20 min at
4°C, the resulting pellet was resuspended in fresh buffer (30 volumes/weight) by
using a vortex. The suspension was incubated for 30 min at 37 °C to remove any
endogenous opioids. Centrifugation was repeated under the same conditions as
described above, and final pellet was resuspended in five volumes of 50 mM Tris–
HCl (pH 7.4) buffer containing 0.32 M sucrose to give a final concentration of 3–4
mg/ml protein. The presence of sucrose is necessary for stabilization of proteins for
storage. The membranes were kept in 5 ml aliquots at -70 °C until use. The binding
activity of the protein remained stable for at least 2 months. Membranes were thawed
and resuspended in 50 mM Tris–HCl (pH 7.4) buffer and centrifuged at 40,000xg
(18,000 rpm) for 20 min at 4 °C to remove the sucrose. The resulting pellets were
taken up in appropriate fresh buffer and immediately used in binding assays. The
protein concentration was determined according to the method of Bradford (1976),
using bovine serum albumin as a standard.
6. Preparation of hNOP-CHO cells
Chinese hamster ovary (CHO) cells stably expressing the wild type human nociceptin
(hNOP) receptor protein were kindly provided by Dr Jean-Claude Meunier, Toulouse,
France. Cells were cultured in a medium containing Nut Mix F-12 with l-glutamine
(GIBCOInvitrogen) and 25 mM Hepes, 10 % FCS, 100 UI/ml penicillin, 100 µg/ml
streptomycin and 0.4 mg/ml G418 at 37ºC in a humidified atmosphere consisting of 5
% CO2 and 95 % air. Cells were sub-cultured twice a week. Cells were harvested with
trypsin solution (0.05 % trypsin, 0.02 % EDTA) in ice cold PBS by washing two
times, frozen at -70°C for 2 hours to facilitate cell disruption by water crystallization
and homogenized in 50 mM Tris-HCl (pH 7.4) buffer with a glass/glass hand
homogenizer (Wheaton USA). Then centrifuged at 1000 x g for 10 min at 4ºC and the
collected pellets were homogenized in 50 mM Tris-HCl (pH 7.4) buffer with a glass /
glass hand homogenizer. Homogenates were centrifuged two times at 40,000 × g for
20 min at 4ºC. Pellets were suspended in Tris-HCl (pH 7.4) buffer and following the
18
protein determination the membrane preparation was aliquoted (0.3-0.4 mg/ml protein
per series) and stored at -70ºC until use. Membranes for the GTPS binding assays
were prepared by the same procedure, but the pellets were suspended in TEM (50 mM
Tris, 1 mM EGTA, 5 mM MgCl2) pH 7.4 buffer. Membrane fractions were aliquoted
(~ 240 µg protein per series = ~ 10 µg protein/sample) and stored at -70ºC until use.
7. Receptor binding assays
All binding assays were performed at 25°C for 30 minutes in 50 mM Tris–HCl buffer
(pH 7.4) in a final volume of 1 ml, containing 1 mg BSA and 0.2–0.4 mg/ml
membrane protein. Samples were made in disposable plastic assay tubes (Sarstedt
Co., Nümbrecht, Germany). Rat and Guinea brain membranes were incubated with
the general opioid agonist radioligand [3H]naloxone (0.8–1.2 nM), or with type-
specific radioprobes, such as the selective MOP receptor agonist [3H]DAMGO (0.9–
1.2 nM), the DOP receptor selective agonist [3H]Tyr1,Ile5,6deltorphin-2 (Nevin et al.,
1994) (Tyr-D-Ala-Phe-Glu-Ile-Ile-Gly-NH2; 0.8–1.3 nM), or with 0.4–0.7 nM of the
KOP receptor-specific agonist [3H]D-Ala3-dynorphin-[1-11]amide and [3H]U-69,593
(with guinea pig brain membranes) and NOP receptor radioprobes [3H]Ac-RYYRIK-
ol and [Leucyl-3H]nociceptin in the presence of unlabeled test ligands (their
concentrations ranged from 10-5 to 10-11 M). Conditions for incubations are given in
the figure legends. Non-specific binding was determined in the presence of 10 M
naloxone for opioid and 10 M N/OFQ for the NOP receptor studies. Reaction was
terminated and bound and free radioligands were separated by rapid filtration under
vacuum through Whatman GF/C (radiolabeled peptides) or GF/B (radiolabeled
alkaloids) glass fiber filters by using Brandel M24R Cell Harvester. Filters were
washed three times with 5 ml ice-cold 50 mM Tris–HCl buffer. After filtration and
separation procedure had been completed, fiber-disks were dried under an infrared
lamp, and then removed from the filter-sheet by tweezers. Each disk was inserted into
UltimaGold™ environment friendly, non-volatile, toluene-free scintillation cocktail,
and placed into individual sample vials (transparent glass, Packard). Bound
radioactivity was determined in Packard Tricarb 2300TR liquid scintillation analyzer.
Receptor binding experiments were performed in duplicate and repeated at least three
times.
19
8. [35S]GTPS binding assays
Rat brain membrane fractions (~10 µg of protein/sample) were incubated at 30°C for
60 min in Tris–EGTA buffer (50 mM Tris–HCl, 1 mM EGTA, 3 mM MgCl2, 100
mM NaCl, pH 7.4) containing [35S]GTPγS (0.05 nM) and increasing concentrations
(10-9 to 10-5 M) of the compounds tested in the presence of 30 µM GDP in a final
volume of 1 ml. Total binding was measured in the absence of test compound, non-
specific binding was determined in the presence of 10 µM unlabeled GTPγS and
subtracted from total binding to calculate the specific binding. The reaction was
started by addition of [35S]GTPγS and terminated by filtrating the samples through
Whatman GF/B glass fiber filters. Filters were washed three times with ice-cold 50
mM Tris–HCl buffer (pH 7.4) using Brandel M24R Cell Harvester, then dried, and
bound radioactivity was detected in UltimaGold™ scintillation cocktail (Packard).
Agonist-induced receptor-mediated G-protein stimulation is given as percentage over
the specific [35S]GTPγS binding observed in the absence of receptor ligands (basal
activity).
9. Isolated tissue bioassay
Tissues for in-vitro studies were taken from male Swiss mice (25–30 g), guinea pigs
(300–350 g), and Sprague Dawley rats (300–350 g). The mouse vas deferens, the rat
vas deferens and the guinea pig ileum was prepared according to Bigoni et al., (1999).
The tissues were suspended in 5-ml organ baths containing Krebs solution oxygenated
with 95% O2 and 5% CO2. The temperature was set at 33°C for the mouse vas
deferens and at 37°C for the other tissues. A resting tension of 0.3 g was applied to
the mouse vas deferens, 1 g to the guinea pig ileum and rat vas deferens. For the
experiments on the mouse vas deferens, an Mg2+-free and a 2.5 mM CaCl2, rat vas
deferens 1.2 mM MgSO4 and a 1.8 mM CaCl2 Krebs solution were used, respectively.
All tissues were continuously stimulated through two platinum ring electrodes with
supramaximal voltage rectangular pulses of 1 ms duration and 0.1 Hz frequency. The
electrically evoked contractions (twitches) were measured isotonically with a strain
gauge transducer (Basile 7006) and recorded with the PC-based acquisition system
Power Lab (ADInstrument, USA). After an equilibration period of about 60 min, the
contractions induced by electrical field stimulation were stable; at this time,
cumulative concentration-response curves to agonists were performed (0.5 log-unit
20
step) in the absence or presence (15 min preincubation time) of antagonists. In these
preparations, the effects of agonists were expressed as percent inhibition of the control
twitch.
For the mouse colon bioassay, approximately 1 cm length segments of mouse colon
were prepared to record the isometric smooth muscle contraction as described
previously (Rizzi et al., 1999). Briefly, the preparations were mounted vertically
under 1 g tension in an organ bath (10 ml) containing Krebs buffer at 37°C and
continuously gassed with 5% CO2 and 95% O2. Tissues were equilibrated for 60 min
with washing every 10 min. For recording the maximal contractile response of the
tissues 100 µM carbachol (carbamylcholine chloride) was used. In the mouse colon,
N/OFQ and Ac-RYYRIR-ol elicit concentration-dependent contractions;
concentration-response curves to these peptides were performed non-cumulatively,
adding to the bath different concentrations of peptide at 20-min intervals. Contractile
effects of N/OFQ or Ac-RYYRIR-ol, were expressed as % of the contraction induced
by carbachol 100 µM.
10. Calcium-ion mobilization experiments
Cultured chinese hamster ovary (CHO) cells stably co-expressing the human
recombinant NOP receptor and the chimeric protein Gqi5 (Camarda et al., 2009)
were maintained in Dulbecco’s Medium (DMEM) supplemented with 10% fetal
bovine serum, 2 mM L-glutamine and 100 mg/L hygromycin and cultured at 37°C in
5% CO2 humidified air. Cells were seeded at a density of 50,000 cells/well into poly
D-lysine coated, 96-well, black, and clear-bottom plates. The following day, the cells
were incubated with medium supplemented with 2.5 mM of probenecid, 3 µM of the
calcium sensitive fluorescent dye Fluo-4 AM, and 0.01% pluronic acid for 30 min at
37°C. After that time, the loading solution was aspirated, and 100 µL/well of assay
buffer (Hank’s balanced salt solution; HBSS) supplemented with 20 mmol of 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2.5 mM of probenecid, and
500 µM of Brilliant Black (Aldrich) were added. Concentrated solutions (1 mmol) of
N/OFQ and Ac-RYYRIR-ol were made in bidistilled water and kept at -20 °C. Serial
dilutions were carried out in HBSS/HEPES (20 mM) buffer (containing 0.02% BSA
fraction V) in order to prepare a master plate at 3× concentration. After placing both
plates (cell culture and master plate) into the fluorometric imaging plate reader
21
FlexStation II (Molecular Devices, Sunnyvale, CA), fluorescence changes were
measured at room temperature ( 22°C). On-line additions were carried out in a
volume of 50 µL/well. Maximum change in fluorescence, expressed as percent over
the baseline fluorescence, was used to determine agonist response.
11. Internalization of the fluorescent receptor
The recombinant HEK293 cells (which were obtained from Dr. Maithe Corbani
Montpellier, France) were harvested using trypsin solution (0.05 % trypsin, 0.02 %
EDTA). Cells (numbers of 30,000 - 50,000) were seeded in glass bottom flask in a 2-
ml volume. The next day, the cells were incubated in 1 ml of Nut Mix F-12 (HAM)
with l-glutamine (GIBCOInvitrogen) culture medium containing 0.1 mg BSA, 25 mm
HEPES (pH 7.4) and the ligand at the desired concentration. Olympus Cell-R
fluorescence microscope was used with GFP filter set combination and 20 x
objectives (37ºC).
12. Data analysis and terminology
All data are expressed as means ± standard error of the mean of n experiments. Curve
fitting was performed using PRISM 4.0 (GraphPad Software Inc., San Diego, U.S.A.).
Agonist potencies were expressed as pEC50, which is the negative logarithm to base
10 of the agonist molar concentration that produces 50% of the maximal possible
effect of that agonist. The Emax is the maximal effect that an agonist can elicit in a
given tissue/preparation. All radioligand binding experiments were performed in
duplicates and the [35S]GTPγS binding assays were performed in triplicates.
Displacement curves were fitted by non-linear regression using the one-site
competition fitting option with no ligand depletion. G-protein stimulation data were
analyzed by the sigmoid dose-response curve fit option of Prism. The equilibrium
inhibition constant (Ki value) was calculated from the IC50 values according to the
built-in Cheng-Prusoff (1973) equation module. Whereas the IC50 value for a
compound may vary between experiments depending on radioligand concentration,
the Ki is an absolute value:
d
i
KLK ][1
IC50
22
where [L] is the concentration of free radioligand used in the assay and Kd is the
dissociation constant of the radioligand for the receptor.
It is known that partial agonists can antagonize the stimulatory effect caused by the
full agonists. In the [35S]GTPγS binding inhibition experiments the antagonist
potency, expressed as pKB, was derived from the following equation:
11
50
50
][2nn
B
ECA
ICpK
where IC50 is the concentration of antagonist that produces 50% inhibition of the
agonist response, [A] is the concentration of agonist, EC50 is the concentration of
agonist producing 50% of the maximal response, and n is the Hill coefficient of the
concentration response curve to the agonist (Kenakin, 2004).
In the assays of the electrically stimulated mouse vas deferens, rat vas deferens and
guinea pig ileum, the Emax of agonists is expressed as % of inhibition of the control
twitch, while for mouse colon data as percent of the contraction elicited by 100 µM
carbachol. Antagonist potencies were expressed as pA2 which is the negative
logarithm to the base 10 of the antagonist molar concentration that makes it necessary
to double the agonist concentration to elicit the original submaximal response
(Arunlakshana and Schild, 1959) and were calculated by Schild's linear regression.
23
RESULTS
NATURALLY OCCURRING NOVEL OPIOID PEPTIDES
1. Bioinformatic examination of the opioid motifs in PDYN and PENK
precursor polypeptides of various species
An analysis of public protein databases (NCBI Protein, Ensembl) has shown
significant diversity in the amino acid sequences of prodynorphin and proenkephalin-
derived opioid peptides. Altogether 39 PDYN sequences have been collected by
database searching. Non-allelic PDYN gene duplicates have been identified in the
tetraploid Xenopus laevis (Pattee et al., 2003). Mammalian PDYN end-products are
identical at the amino acid level with the exception of the C-terminal tail region of
dynorphin B. Organization and peptide sequence variability appears more complex
among PDYNs, since both the number and the length of the opioid units are rather
different within this precursor. The C-terminal section of PDYNs that contains -
neo-endorphin, dynorphin A and dynorphin B (also termed as rimorphin) is notably
conserved, while the N-terminal segment of the non-mammalian PDYN precursors is
substantially flexible in terms of amino acid composition. A common feature of these
PDYNs is the presence of at least one or even more opioid core motif located
upstream relative to the position of the ‘opioid triad’ in mammalian PDYNs. Thus,
four enkephalin units are present in the PDYN of the cane toad Bufo marinus, the
zebrafish Danio rerio, and three other fish species. The PDYNs of the Australian
lungfish (Neoceratodus forsteri), and two frog species including Bombina orientalis
and Xenopus laevis contain five opioid repeats. The recently identified Ile-enkephalin
lies in identical position amongst the PDYN orthologs. The unique Phe-enkephalin
pentapeptide is located in the second place within the partially sequenced PDYN of
the African lungfish (Protopterus annectens). Based on structural information,
PDYNs might be biosynthetic sources for at least four kinds of enkephalins in some
species. Frogs appear quite unusual animals in this context, because frog PENKs
include only Met-enkephalin repeats, whereas PDYNs of certain frog species contain
an atypical Met5-dynorphin A, thus suggesting a further, non proenkephalin derived
source of Met-enkephalin in these amphibians. Fish dynorphin A is seemingly a
24
Met5-dynorphin in each species studied so far, although fish -neo-endorphin and
dynorphin B sequences contain the conventional Leu-enkephalin units.
Fig. 4. Alignment of human, mouse, chicken, xenopus, lungfish and shark proenkephalin sequences. Human (Homo sapiens; NP_001129162), Mouse (Mus musculus; NP_001002927), Chicken (Gallus gallus; XP_419213), Frog (Xenopus laevis; AAB20686 and Xenopus tropicalis; ENSXETP00000012480), Bombina (Bombina orientalis; AAU95755) Lungfish (Neoceratodus forsteri; AAF44658 and Protopterus annectens; AAF44657) and Shark (Heterodontus portusjacksoni; AAU95733). The opioid positions are labelled and sequences are bold and underlined. Modified amino acid positions in comparison with humans are coloured with red. Endoproteolytic cleavage sites are coloured with blue. Only 100% identical amino acid positions are shaded in gray.
25
Fourteen distinct orthologues have been identified in the case of PENK derived
octapeptides amongst 55 species. The ubiquitous YGGFMRGL sequence is present
in 26 mammals including the human, although more other mammalian octapeptides
exist, e.g., YGGFMRGY (Xenopus laevis, Ornithorhyncus anatinus), YGGFMRSY
(rainforest opossum, Monodelphis domestica), YGGFMRAL (cat and dog),
YGGFMRSL (mouse), YGGFMRGV (European rabbit, Oryctolagus cuniculus and
American pika, Ochotona princeps). Of the other vertebrate animals tested, including
ten fish species, seven amphibians, four reptiles and two birds, even more variability
in the structure of the octapeptide is observed (due to space limitation, alignments of
all available PENK and PDYN sequences are not shown).
Fig. 5 Evolution scheme of the major groups within superclass Gnathostomata, exhibiting various PENK and PDYN sequences studied here. Nomenclature: vertebrates (subphylum Vertebrata), gnathostomes (jawed animals), actinopterygians (class Actinopterygii), sarcopterygians (class Sarcopterygii), tetrapods (subclass Tetrapoda), amniotes (subclass Amniota). The phylogenetic three is adapted from Dores et al., (2002).
Four different sequences have been selected for the chemical synthesis: The ‘mouse
sequence’ YGGFMRSL (1) is unique among mammals, but is also present in the
Australian lungfish (Neoceratodus forsteri) and African lungfish (Protopterus
annectens) proenkephalins (Sollars et al., 2000; Dores et al., 2001). The double
substituted YGGFMNGF (2) sequence distinctively found in a cartilaginous fish
species (Komorowski et al., 2004), the bullhead shark (Heterodontus portusjacksoni),
is the only one in that the Arg6 residue is replaced (R→N). Moreover, the shark
octapeptide ends with an aromatic phenylalanine (Phe8) side chain as a consequence
of L→F substitution. The structure of YGGFMRGY (3) represents another
26
orthologous peptide bearing aromatic (Tyr8) C-terminus due to L→Y replacement. In
addition to the platypus, this sequence also occurs in two pipide frog species, the
tetraploid Xenopus laevis (Martens and Herbert, 1994) and the diploid Xenopus
tropicalis, and in the fire-bellied toad, Bombina orientalis (Dores et al., 2001). The
YGGFMRSV (4) octapeptide is included in the chicken proenkephalin (prediction
from genomic sequence, NCBI), moreover in the incompletely sequenced PENK of
the florida gar (Lepisosteus platyrhynchus) fish species (Khalap et al., 2005). All full-
size propeptides carrying various octapeptide components can well be aligned to the
human sequence (Fig. 4).
2. Receptor binding assays
The mammalian and non-mammalian enkephalins and octapeptides were first tested
in [3H]naloxone binding assays. Naloxone is an opioid antagonist with high affinity
and capable of interacting with all types of opioid receptors. Each novel analogue
displayed moderate, and more or less similar affinity in heterologous competition
experiments with [3H]naloxone, while the homologous ligand unlabelled naloxone
exhibited the highest affinity in these experiments (Fig. 6, left panel; Fig. 7).
-11 -10 -9 -8 -7 -6 -5Total
0
20
40
60
80
100
Leu-enkephalin
NaloxonePhe-enkephalinIle-enkephalin
Met-enkephalin
Non-selective assay
log[Ligand], (M)
% [3 H
]Nal
oxon
e s
peci
fic b
indi
ng
-11 -10 -9 -8 -7 -6 -5Total
0
25
50
75
100
Ile-enkephalinPhe-enkephalin
Leu-enkephalinMet-enkephalin
DAMGO
MOPreceptor selective assay
log[Ligand], (M)
% [3 H
]DA
MG
Osp
ecifi
c bi
ndin
g
Fig. 6. Equilibrium competition binding of enkephalins at the MOP receptor sites. Left panel: [3H]naloxone binding (0.8-1.2 nM, 60 mins, 0-4°C); Right panel: [3H]DAMGO binding (0.9-1.2 nM, 45 mins, 24°C); Points represent the means SEM of at least three different experiments each performed in duplicate.
Using a MOP receptor specific radioprobe, the peptide [3H]DAMGO, all of the novel
sequences could displace the radiprobe in concentration deppendent manner, with
homologous unlabelled ligand DAMGO displaying the best affinity. Lungfish Phe-
27
enkephalin, and frog Ile-enkephalin displaced effectively the bound radioligand from
the receptor (Fig. 6 right panel).
-12 -11 -10 -9 -8 -7 -6 -5 -40
20
40
60
80
100
NaloxoneYGGFMRGYYGGFM(O)RGYYGGFMRGL
log[Ligand], (M)-12 -11 -10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100
NaloxoneYGGFMRSVYGGFM(O)RSV
log[Ligand], (M)
-12 -11 -10 -9 -8 -7 -6 -5 -40
20
40
60
80
100
NaloxoneYGGFMNGFYGGFM(O)NGFMet enkephalin
log[Ligand], (M)-12 -11 -10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100
NaloxoneYGGFMRSLYGGFM(O)RSL
log[Ligand], (M)
%
[3 H]N
alox
one
spe
cific
bin
ding
Fig. 7. Equilibrium competition binding of octapeptides at the non selective opioid receptor sites, using [3H]naloxone. (0.6-1.1 nM, 60 mins, 0-4°C); Points represent the means SEM of at least three different experiments each performed in duplicate.
In the case of octapeptides, YGGFMRSL (mouse) and YGGFMRSV (chicken) were
most effective while YGGFMRGY (frog) and YGGFMNGF (shark) were weaker for
activation of the MOP receptor under the conditions used, meaning that the novel
peptides had relatively good or moderate potencies in competing reversibly with the
MOP binding sites under the conditions used (Fig. 8).
Binding properties of the compounds at the DOP receptors were measured with a
radiolabelled frog skin peptide ligand analogue, Ile5,6deltorphin-2 ([3H]Tyr-D-Ala-
Phe-Glu-Ile-Ile-Gly-NH2). Although mammalian enkephalins are preferentially DOP
receptor selective compounds, yet in our assays both the lungfish and frog
enkephalins failed to effectively compete for the labelled deltorphin sites, therefore
Ile- and Phe- enkephalins turned out to be weaker ligands at the opioid δ-sites (Fig.
10, left panel). The rank order of potency was Ile5,6deltorphin-2 > Met-enkephalin >
Leu-enkephalin > Ile-enkephalin > Phe-enkephalin.
28
-12 -11 -10 -9 -8 -7 -6 -5 -40
20
40
60
80
100
YGGFMRGYYGGFM(O)RGY
DAMGO
YGGFMRGL
log[Ligand], (M)-12 -11 -10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100
YGGFMRSVYGGFM(O)RSV
DAMGO
log[Ligand], (M)
-12 -11 -10 -9 -8 -7 -6 -5 -40
20
40
60
80
100
YGGFMNGFYGGFM(O)NGF
DAMGO
Met-enkephalin
log[Ligand], (M)-12 -11 -10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100
YGGFMRSLYGGFM(O)RSL
DAMGO
log[Ligand], (M)
% [3 H
]DA
MG
O s
peci
fic b
indi
ng
Fig. 8. Equilibrium competition binding of octapeptides at the MOP receptor sites, using [3H]DAMGO. (0.7-1.1 nM, 45 mins, 24°C); Points represent the means SEM of at least three different experiments each performed in duplicate.
-12 -11 -10 -9 -8 -7 -6 -5 -40
20
40
60
80
100
YGGFMRGYYGGFM(O)RGY
DIDI
YGGFMRGL
log[Ligand], (M)-12 -11 -10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100
YGGFMRSVYGGFM(O)RSV
DIDI
log[Ligand], (M)
-12 -11 -10 -9 -8 -7 -6 -5 -40
20
40
60
80
100
YGGFMNGFYGGFM(O)NGF
DIDI
Met-enkephalin
log[Ligand], (M)-12 -11 -10 -9 -8 -7 -6 -5 -4
0
20
40
60
80
100
YGGFMRSLYGGFM(O)RSL
DIDI
log[Ligand], (M)
% [3 H
]Ile5,
6 -del
torp
hin-
2 sp
ecifi
c bi
ndin
g
Fig. 9. Equilibrium competition binding of octapeptides at the DOP receptor sites, using [3H]Ile5,6deltorphin-2. (0.9-1.4 nM, 45 mins, 24°C); Points represent the means SEM of at least three different experiments each performed in duplicate.
29
Octapeptides sequences similar to the MOP assay could displace the
[3H][3H]Tyr1,Ile5,6-deltorphin-2 radiprobe in a concentration dependent manner from
the opioid δ-sites. Again, YGGFMRGY (Frog) and YGGFMNGF (Shark) were less
potent, while YGGFMRSV (chicken) and YGGFMRSL (mouse) were more effective
in the sense of DOP receptor activation (Fig. 9).
-11 -10 -9 -8 -7 -6 -5Total
0
20
40
60
80
100
120
Met-enkephalin
Ile-enkephalinPhe-enkephalin
Leu-enkephalin
Ile5,6deltorphin-2
DOP receptor selective assay
log[Ligand], (M)
% [
3 H]Il
e5,6 de
ltorp
hin-
2sp
ecifi
c bi
ndin
g
-11 -10 -9 -8 -7 -6 -5Total
0
20
40
60
80
100
120
Leu-enkephalin
Dynorphin-[1-11]
Ile-enkephalinPhe-enkephalin
Met-enkephalin
KOP receptor selective assay
log[Ligand], (M) %
[3 H
]D-A
la3 -D
ynor
phin
-[1
-11]
amid
e s
peci
fic b
indi
ng
Fig. 10 . Equilibrium competition binding of enkephalins at the DOP and KOP receptor sites. Left panel: [3H]Ile5,6deltorphin-2 binding (0.8-1.3 nM, 45 mins, 24°C); Right panel: [3H]D-Ala3-dynorphin-[1-11]amide binding (0.4-0.7 nM, 30 mins, 24°C) Points represent the means SEM of at least three different experiments each performed in duplicate.
Enkephalin analogues were further studied at the KOP receptor sites labelled by a
tritiated synthetic dynorphin A analogue, D-Ala3-dynorphin[1-11] (Lung et al., 1995).
Predictably all enkephalins were less potent in this assays comparing with the
affinities observed in the DOP receptor sensitive assays (Fig. 10, right panel).
Nevertheless, the affinity of Phe-enkephalin was the best in this KOP receptor
selective assay system. The other enkephalins investigated here were roughly
equipotent, but the experimental errors were higher in this assay. This is likely due to
the enzymatic sensitivity and adsorptive properties of dynorphin peptides, although
several endopeptidase inhibitors, such as EDTA, trasylol, bacitracin, and
phenylmethylsulfonyl fluoride (PMSF) were also included in the assays.
On the other hand, KOP receptor selectivity of octapeptides were studied with a
nonpeptide benzeneacetamide compound, U-69,593 which is among the most potent
and selective ligand for the specific labelling of KOP receptors (Rahmani et al.,
1991). In guinea pig membranes none of the octapeptide sequences displayed any
affinity in this system. Opioid activity of all these peptides was inactive in the KOP
system (Fig. 11).
30
-12 -11 -10 -9 -8 -7 -6 -5 -40
25
50
75
100
YGGFMRGYYGGFM(O)RGY
U-69
YGGFMRGL
log[Ligand], (M)-12 -11 -10 -9 -8 -7 -6 -5 -4
0
25
50
75
100
YGGFMRSVYGGFM(O)RSV
U-69
log[Ligand], (M)
-12 -11 -10 -9 -8 -7 -6 -5 -40
25
50
75
100
YGGFMNGFYGGFM(O)NGF
U-69
Met-enkephalin
log[Ligand], (M)-12 -11 -10 -9 -8 -7 -6 -5 -4
0
25
50
75
100
YGGFMRSLYGGFM(O)RSL
U-69
log[Ligand], (M)
[3 H]U
69,5
93 s
peci
fic b
indi
ng
Fig. 11 . Equilibrium competition binding of octapeptides at the KOP receptor sites, using [3H]U-69,593. (0.6-1.1 nM, 60 mins, 24°C); Points represent the means SEM of at least three different experiments each performed in duplicate.
The oxidation of the Met5 residue reduced the affinity of the enkephalins by shifting
the competition curves to the right. In each receptor binding assay, all the oxidized
form of the octapeptides was less potent (Figs 7-8-9-11).
Table 4 is summarizing the binding data of the novel opioid sequences studied,
together with the relative affinities of the peptides towards the opioid receptors.
31
Ligand Equilibrium affinity logIC50±S.E.M. (Ki, nM)a
Non-selective DOP assay, δ
MOP assay, µ
KOP assay, κ
% Relative affinityb δ: μ: κ
Naloxone 8.62±0.07 (1.54) - - - -
DIDI - 8.55±0.07 (1.92) - - -
DAMGO - - 8.72±0.04 (1.28) - -
U-69,593 - - - 8.33±0.05 (4.61) -
Dynorphin-[1-11] - - - 8.51±0.12 (2.73) -
Met-enkephalin 7.74±0.08 (11) 7.91±0.08 (8.6) 7.52±0.08 (20) 5.86±0.5 (1117) 69:30:1
Ile-enkephalin (Frog) 7.20±0.1 (29) 6.92±0.1 (20.8) 6.45±0.07 (211) 6.880.2± (125) 79:8:13
Phe-enkepahlin (Fish) 6.98±0.1 (52) 6.19±0.1 (111) 6.14±0.06 (432) 7.49±0.2 (30.4) 20:6:74
YGGFMRGL (Human) 7.82±0.04 (9.9) 7.88±0.09 (9.1) 7.58±0.06 (17) 6.48±0.09 (269) 64:34:2
YGGFMRGY (Frog) 6.87±0.1 (87) 6.82±0.08 (104) 6.87±0.07 (95) 5.57±0.1 (2211) 47:51:2
YGGFM(O)RGY 6.47±0.1 (213) 6.42±0.08 (258) 6.37±0.1 (258) 5.38±0.3 (3363) 51:45:4
YGGFMRSV (Chicken) 7.76±0.06 (11) 7.62±0.08 (16) 7.65±0.08 (15) 5.41±0.1 (3204) 48:51:1
YGGFM(O)RSV 7.62±0.01 (15) 6.92±0.07 (81) 7.29±0.08 (34) 5.67±0.1 (1763) 30:69:1
YGGFMNGF (Shark) 7.74±0.07 (12) 7.15±0.05 (48) 7.05±0.05 (59) 5.74±0.3 (1494) 54:44:2
YGGFM(O)NGF 7.19±0.06 (41) 6.93±0.06 (79) 6.55±0.07 (189) 5.87±0.4 (1137) 67:28:5
YGGFMRSL (Fish) 7.11±0.04 (49) 7.11±0.07 (45) 7.78±0.06 (14) 5.55±0.2 (2285) 24:75:1
YGGFM(O)RSL 7.04±0.06 (58) 6.93±0.06 (81) 7.01±0.06 (67) 5.44±0.2 (2952) 45:54:1
a Calculated by the Chenc-Prusoff equation b % Relative affinity calculated according to Kosterlitz and Paterson, 1980 [% Relative affinity={(1/Ki)/(1/Ki+1/Ki+1/Ki)}x100] Table 4. Completion of the novel opioid sequences in a set of opioid receptor type-selective radioligand binding assays in brain membrane fractions
32
3. [35S]GTPγS binding experiments
[35S]GTPγS binding assays represent functional tests determining agonist-induced,
receptor-mediated G-protein activation. The assay is based on the increase in guanine
nucleotide exchange at G-proteins upon agonist stimulation. All enkephalins
effectively stimulated the G-proteins on rat brain membranes indicating the agonist
properties of the ligands tested (Fig. 12). In terms of maximal stimulation, Met-
enkephalin caused the highest effect, but all other peptides were also decidedly
effective.
-10 -9 -8 -7 -6 -5Basal
90
115
140
165
190
215
240Met-enkephalinLeu-enkephalinIle-enkephalinPhe-enkephalin
log[Ligand], (M)
% o
f GTP
[35S]
bin
ding
over
the
basa
l lev
el
Fig. 12. Agonist-induced activation of G-proteins in rat brain membranes. Stimulation is given as a percentage of the nonstimulated (basal) level. Basal activity (basal = ‘total binding’ – ‘nonspecific binding’) is taken as 100%. Nonspecific binding is determined in the presence of 10 µM unlabeled GTPγS. Incubations were carried out with 0.05 nM [35S]GTPS for 60 mins at 30°C with gentle shaking. Points represent the means SEM of at least three independent determinations each performed in triplicate.
Novel octapeptides as well effectively stimulated the G-proteins on rat brain
membranes (Fig. 13). In terms of maximal stimulation, again Met-enkephalin caused
the highest effect, but all other octapeptide analogues were also very effective in G-
protein activation. While potency values (pEC50) were higest in YGGFMRSV and
YGGFMRSL, maximum stimulation (efficacy, %Emax) was relatively similar in all the
peptides used for the [35S]GTPγS binding assays. Oxidized analogues of the
octapeptides were again less efficient for G-protein activation. The maximal
stimulation levels (efficacy, %Emax) and the potency (pEC50) values of the ligands are
summarized in Table 5.
33
-11 -10 -9 -8 -7 -6 -5 -4
100
130
160
190
220
YGGFMRGYYGGFM(O)RGY
Met-enkephalin
YGGFMRGL
log[Ligand], (M)-11 -10 -9 -8 -7 -6 -5 -4
100
130
160
190
220
YGGFMRSVYGGFM(O)RSV
Met-enkephalin
log[Ligand], (M)
-11 -10 -9 -8 -7 -6 -5 -4
100
130
160
190
220
YGGFMNGFYGGFM(O)NGF
Met-enkephalin
Leu-enkephalin
log[Ligand], (M)-11 -10 -9 -8 -7 -6 -5 -4
100
130
160
190
220
YGGFMRSLYGGFM(O)RSL
Met-enkephalin
log[Ligand], (M)% s
timul
atio
n of
[35S]
GTP
S b
indi
ng o
ver t
he b
asal
Fig. 13. Agonist-induced activation of G-proteins in rat brain membranes. Nonspecific binding is determined in the presence of 10 µM unlabeled GTPγS. Incubations were carried out with 0.05 nM [35S]GTPS for 60 mins at 30°C with gentle shaking. Points represent the means SEM of at least three independent determinations each performed in triplicate.
Peptides
Potency logEC50±S.E.M
(EC50, nM)
Efficacy (Emax), % stimulation ±S.E.M
over the basal Met-enkephalin 7.35±0.08 (60) 234±3.8
Leu-enkephalin 6.84±0.08 (145) 178±6
Ile-enkephalin 6.63±0.23 (237) 176±18
Phe-enkephalin 6.49±0.11 (323) 168±19
YGGFMRGL (Human) 6.22±0.07 (594) 182±2.8
YGGFMRGY (Frog) 6.03±0.12 (993) 194±6.3
YGGFM(O)RGY 5.7±0.08 (1990) 186±4.6
YGGFMRSV (Chicken) 6.28±0.09 (515) 191±4.1
YGGFM(O)RSV 5.6±0.6 (2465) 197±4.0
YGGFMNGF (Shark) 5.87±0.08 (1335) 207±5.4
YGGFM(O)NGF 5.4±0.1 (3958) 203±8.2
YGGFMRSL (Fish) 6.18±0.08 (653) 220±5.5
YGGFM(O)RSL 5.7±0.12 (1979) 226±9.7
Table 5. G-protein activation by opioid peptides in the rat brain membranes
34
IN-VITRO PHARMACOLOGICAL CHARACTERIZATION OF A NOVEL,
NOP RECEPTOR SELECTIVE HEXAPEPTIDE, Ac-RYYRIR-ol
1. Receptor binding assays
The radioligand binding studies were performed with [3H]Ac-RYYRIK-ol using rat
brain membranes. Binding studies were done using N/OFQ and the hexapeptide
alcohols Ac-RYYRIK-ol, Ac-RYYRIR-ol. All three peptides displaced [3H]Ac-
RYYRIK-ol in a concentration dependent manner. Ac-RYYRIR-ol was relatively the
weakest and Ac-RYYRIK-ol showed the highest affinity to the NOP receptor in the
competition binding experiments (Fig. 14). The pKi values were as follows; Ac-
RYYRIK-ol 8.85, N/OFQ 8.69 and Ac-RYYRIR-ol 8.26.
-12 -11 -10 -9 -8 -7 -6 -50
20
40
60
80
100
Ac-RYYRIK-olN/OFQAc-RYYRIR-ol
log[Ligand], M
[3 H]A
c-R
YYR
IK-o
l,sp
ecifi
c bi
ndin
g %
Fig. 14. Equilibrium competition binding of the Ac-RYYRIK-ol, N/OFQ and Ac-RYYRIR-ol at NOP receptor using [3H]Ac-RYYRIK-ol in rat brain membranes. The nonspecific binding was measured with 10 M Ac-RYYRIK-ol. Data are means ±SEM of 3 experiments performed in duplicate.
Receptor binding experiments done with [3H]DAMGO and [3H]Ile5,6deltorphin-II
revealed that Ac-RYYRIR-ol has no any affinity toward these opioid receptors. The
novel hexapeptide could not displace both radioprobes, even up to very high
concentrations (i.e. 10 µM, data not shown).
2. [35S]GTPγS binding assays
In the [35S]GTPγS binding assays N/OFQ effectively stimulated G-proteins in rat
brain membranes (Emax 64 ± 2%, pEC50 7.04) defining the full agonist effect at the
NOP receptors (Fig. 15, left panel). Ac-RYYRIR-ol could also stimulate the G-
protein activation with similar potency (pEC50 6.73) but far less efficacy (Emax 18 ±2
35
%, = 0.28) compared to N/OFQ (Fig. 15, left panel). Stimulatory effect of both
N/OFQ and Ac-RYYRIR-ol could be inhibited using specific NOP receptor
antagonist UFP-101 (Figure 15, left panel) and pA2 value of 6.9 vas calculated. In the
inhibition experiments, the effects of increasing concentration of Ac-RYYRIR-ol
were tested against the stimulatory effect produced by a fixed concentration of
N/OFQ (1 µM). Ac-RYYRIR-ol effectively inhibited in a concentration dependent
manner the stimulatory effect of 1 µM N/OFQ with a maximal inhibitory effect
similar to that estimated in agonist type experiments. A pKB value of 7.29 was derived
from these experiments (Fig 15 , right panel).
-11 -10 -9 -8 -7 -6 -5
010203040506070
Ac-RYYRIR-ol
N/OFQ
UFP101 1M
+UFP101 1 M
log[Ligand], M
[35S]
GTP
S b
indi
ng%
ove
r th
e ba
sal
-10 -9 -8 -7 -6 -5
010203040506070
Ac-RYYRIR-ol
Ac-RYYRIR-ol+ N/OFQ 1 M
log[Ac-RYYRIR-ol], M
[35S]
GTP
S b
indi
ng%
ove
r th
e ba
sal
Figure 15. Stimulation of [35S]GTPγS binding in rat brain membranes. Left: UFP-101 antagonizing response versus N/OFQ and Ac-RYYRIR-ol, in the [35S]GTPγS binding assay. Data are means ±SEM of 3 experiments performed in triplicate. Right: Inhibition concentration response curves of Ac-RYYRIR-ol against the effects evoked by 1 µM N/OFQ.
In order to study the possible antagonist action of Ac-RYYRIR-ol in a separate series
of experiments using [35S]GTPγS binding assay, the hexapeptide was challenged at
increasing concentration against the concentration response curve to N/OFQ. From
this experiment Ac-RYYRIR-ol potency as an antagonist towards the NOP receptors
has been determined and pA2 was found to be 8.33 with the correlation coefficient of
0.99 (Fig 16).
36
-11 -10 -9 -8 -7 -6 -5
01020304050607080 Control
Ac-RYYRIR-ol 10 nMAc-RYYRIR-ol 100 nMAc-RYYRIR-ol 1 M
log[N/OFQ], M
[35S]
GTP
S b
indi
ng%
ove
r th
e ba
sal
-8 -7 -6
0.2
0.4
0.6
0.8
0
R2 : 0.998
pA2 : 8.33
Slope : 0.33
log[Ac-RYYRIR-ol], M
Log(
DR
-1)
Fig. 16. Stimulation of [35S]GTPγS binding to rat brain membranes. Concentration response curve of N/OFQ, obtained in the absence (control) and presence of increasing concentrations of Ac-RYYRIR-ol (10–1000 nM). On the right panel the corresponding Schild plot is shown. Data are means ±SEM of 3 experiments performed in triplicate.
3. Electrically stimulated isolated tissues and mouse colon bioassay
In the mouse vas deferens, N/OFQ inhibited the twitch response to electrical field
stimulation in a concentration dependent manner showing an Emax of 90±5% and a
pEC50 of 7.60 (Fig. 17, left panel). In this preparation Ac-RYYRIR-ol was completely
inactive at 10 and 100 nM while at 1 µM it produced a slight inhibition of the
electrically induced twitch in some but not all tissues. However, Ac-RYYRIR-ol
tested over the concentration range of 10–1000 nM shifted the concentration response
curve to N/OFQ to the right in a concentration dependent manner. Curves obtained in
the presence of Ac-RYYRIR-ol were parallel to the control and reached similar
maximal effects even in the presence of the highest concentration of compound. The
corresponding Schild plot was linear and slope of the regression line was found to be
0.91 with a pA2 value of 7.99 (Fig. 17, right panel). N/OFQ could also inhibit the
stimulation caused by the electrical impulses in rat vas deferens showing an Emax of
87±2% and a pEC50 of 7.29, while again Ac-RYYRIR-ol was inactive until 1 µM
concentration (Data not shown). In rat vas deferens assay 100 nM Ac-RYYRIR-ol
could inhibit the effect of N/OFQ in these preparations. pA2 value of 8.20 was
calculated for rat vas deferens.
37
-10 -9 -8 -7 -6 -50
20
40
60
80
100
ControlAc-RYYRIR-ol 10 nMAc-RYYRIR-ol 100 nMAc-RYYRIR-ol 1 µM
log[N/OFQ], M
% C
ontr
ol t
witc
h
-8 -7 -6
-0.5
0.0
0.5
1.0
1.5
2.0 pA2 : 7.99R2 : 0.991Slope : 0.91
log[Ac-RYYRIR-ol], M
Log(
CR
-1)
Fig. 17. Inhibitory effect of the hexapeptide in the electrically stimulated mouse vas deferens. Concentration response curve of N/OFQ, obtained in the absence (control) and presence of increasing concentrations of Ac-RYYRIR-ol (10–1000 nM). On the right panel the corresponding Schild plot can be seen. Data are means ±SEM of 3 experiments performed in duplicate.
In the isolated mouse colon, N/OFQ evoked concentration dependent contractile
effects showing a pEC50 of 8.82 and an Emax of 32 ± 4 %. This effect was mimicked
by Ac-RYYRIR-ol which displayed a similar maximal effects (28 ± 3 %) and a
slightly higher value of potency (pEC50 9.09) (Fig. 18).
-11 -10 -9 -8 -7
0
10
20
30
40N/OFQAc-RYYRIR-ol
log[Ligand], M
% C
arba
chol
con
trac
tion
(100
µM
)
Fig. 18. Concentration response curves of N/OFQ and Ac-RYYRIR-ol in the isolated mouse colon. Data are mean ± SEM of 3 experiments performed in duplicate.
In order to examine delta and mu opioid receptor potency of Ac-RYYRIR-ol in the
electrically stimulated tissues, we have studied the concentration response curves to
DPDPE in the mouse vas deferens and to dermorphin in the guinea pig ileum. Curves
were obtained in the absence and presence of a single concentration of Ac-RYYRIR-
ol (1 M). The hexapeptide was found to be completely inactive in both preparations.
38
4. Calcium ion mobilization experiments
Calcium mobilization assays were performed in a fluorometric imaging plate reader
FlexStation II using the CHO cells expressing the recombinant human NOP receptor
and the chimeric protein Gqi5. Results demonstrated that both N/OFQ and Ac-
RYYRIR-ol could stimulate in a concentration-dependent manner calcium
mobilization with pEC50 values of 9.19 and 7.94, and Emax of 240±16% and 161±21%
over the basal levels, respectively (Fig. 19). Thus, Ac-RYYRIR-ol behaved as a
partial agonist, showing a reduction in maximal effects compare to N/OFQ.
-12 -11 -10 -9 -8 -7 -6
0
4080
120160
200240
Ac-RYYRIR-olN/OFQ
log[Ligand], M
FIU
(% o
ver
the
basa
l)
Fig. 19. Concentration response curves of N/OFQ and Ac-RYYRIR-ol in CHO cells expressing the human NOP receptor and the chimeric protein Gqi5. Changes in intracellular Ca2+ ion levels were expressed as % increase of fluorescence intensity units (FIU) over basal. Data are mean ± SEM of 3 experiments performed in duplicate.
5. Internalization of the NOP receptors
Prototype hexapeptide Ac-RYYRIK-ol and novel analogue Ac-RYYRIR-ol was not
efficient to promote NOP receptor internalization in cultured human embryonic
kidney (HEK293) cells expressing the fusion construct hNOP-GFP (green fluorescent
protein), while the full agonist N/OFQ internalized the majority of the receptor within
1 hour (Fig. 20 white arrows).
39
Fig. 20 Olympus Cell-R fluorescence microscope was used with GFP filter set combination and 20 x objectives.
40
SELECTIVE AND HIGH AFFINITY LABELING OF THE NOP RECEPTORS
WITH THE HEXAPEPTIDE RADIOPROBE [3H]Ac-RYYRIK-ol
Heterologous competition experiments with [3H]Ac-RYYRIK-ol
Competition assays were performed using various NOP receptor related ligands such
as the full-size nociceptin (N/OFQ(1-17)), Tyr1-N/OFQ(1-17), the NOP receptor
antagonist peptide UFP-101, the non-peptide antagonist J-113397, several acetyl-
hexapeptides, e.g., Ac-RYYRIK-ol, Ac-RYYRIK-NH2 (parent compound, or
Dooley’s-peptide), the Arg6-derivative Ac-RYYRIR-ol, the citrulline substituted
(Cit4) analogues Ac-RYYCitIK-NH2 and Ac-RYYCitIK-ol (Gündüz et al., 2006-07).
Some opioid compounds including the general opioid receptor antagonist naloxone,
KOP receptor agonist/MOP receptor antagonist buprenorphine, naloxone-
benzoylhydrazone, an antagonist compound known to interact with both KOP and
NOP receptors, the MOP receptor selective peptide agonist [D-Ala2,NMePhe4,Gly5-
ol]enkephalin (DAMGO) and the KOP receptor agonist peptide D-Ala3-dynorphin(1-11)
were also studied to determine their displacing capability in rat brain membranes. All
NOP receptor ligands efficiently displaced [3H]Ac-RYYRIK-ol binding with varying
affinities, in concentration dependent manner (Fig. 21). Opioid compounds exhibited
less potency in competing reversibly for the [3H]Ac-RYYRIK-ol recognition sites,
although moderate affinities were obtained with buprenorphine, naloxone-
benzoylhydrazone and D-Ala3-dynorphin(1-11) (Fig. 21). Rank order of the affinity
was: Ac-RYYRIK-ol > N/OFQ > Tyr1-N/OFQ(1-17) > Ac-RYYRIK-NH2 > Ac-
RYYRIR-ol > UFP-101 > Ac-RYYCitIK-ol > Ac--RYYCitIK-NH2 > J-113397 >
naloxone-benzoylhydrazone > buprenorphine > D-Ala3-dynorphin(1-11) > I2Tyr1-
N/OFQ(1-17)-NH2 > naloxone and DAMGO. The latter two opioids were unable to
produce even 20% inhibition of the specific [3H]Ac-RYYRIK-ol binding. The
equilibrium inhibition constant (Ki) values are summarized in Table 6.
41
-12 -11 -10 -9 -8 -7 -6 -5 -40
20
40
60
80
100
N/OFQAc-RYYRIK-ol
Ac-RYYRIR-ol
Ac-RYY-Cit-IK-NH2
Ac-RYY-Cit-IK-olAc-RYYRIK-NH2
(Tyr1)N/OFQ
log[Ligand], M
[3 H] A
c-R
YYR
IK-o
l,sp
ecifi
c bi
ndin
g %
-12 -11 -10 -9 -8 -7 -6 -5 -40
20
40
60
80
100
Ac-RYYRIK-olUFP 101J-113397Buprenorphine
Naloxone
D-Ala3-Dynorphin1-11
DAMGO
log[Ligand], M
[3 H] A
c-R
YYR
IK-o
l,sp
ecifi
c bi
ndin
g %
Fig. 21 Equilibrium competition binding of various nociceptin (Left Panel) and opioid (Right Panel) ligands at the [3H]Ac-RYYRIK-ol sites in rat brain membranes. [3H]Ac-RYYRIK-ol (0.2–0.4 nM) was incubated in the presence of increasing concentrations of homologous or heterologous competitors, compound names are indicated in the figure. Points represent the means S.E.M. of at least three different experiments each performed in duplicate. Homologous competition curves are shown in both panels for comparison.
[3H]Ac-RYYRIK-ol binding was also studied in cultured Chinese Hamster Ovary
(CHO) cells stably transfected with recombinant human NOP receptor. Competition
studies were performed with [3H]Ac-RYYRIK-ol and [3H]N/OFQ(1–17) (Amersham)
permitting comparisons for the NOP receptor sites labeled by the hexapeptide and
heptadecapeptide radioligands. In homologous competition studies performed on
hNOP-CHO cell membranes almost the same binding affinities were measured for Ac-
RYYRIK-ol either with [3H]Ac-RYYRIK-ol or with [3H](Leu14)nociceptin. Results of
the cross- and self-displacement studies (‘competition tetrad analyses’) are shown in
Fig. 22.
-12 -11 -10 -9 -8 -7 -6 -5
0
20
40
60
80
100
Ac-RYYRIK-olN/OFQ
log[Ligand], M
[3 H] A
c-R
YYR
IK-o
l%
spe
cific
bin
ding
-12 -11 -10 -9 -8 -7 -6 -5
0
20
40
60
80
100
N/OFQAc-RYYRIK-ol
log[Ligand], M
[3 H]N
/OFQ
% s
peci
fic b
indi
ng
Fig. 22. Cross- and self-displacement studies with two radioligands in Chinese Hamster Ovary cells expressing recombinant human nociceptin receptor (hNOP-CHO). (Left Panel) [3H]Ac-RYYRIK-ol binding (0.2–0.3 nM, 30 min, 24º8C). (Right Panel) [3H](Leu14)N/OFQ(1–17) (nociceptin) binding (0.05–0.15 nM, 30 min, 24º8C).
42
Compound
Property
[3H]Ac-RYYRIK-ol pIC50±S.E.M Ki (nM) *
N/OFQ(1-17) NOP agonist (endogenous sequence) 8.56±0.09 2.3
Tyr1-N/OFQ(1-17) Tyr1-substituted analogue 8.50±0.07 2.6
I2Tyr1-N/OFQ(1-17)-NH2 Iodinated analogue 5.04±0.40 7726
Ac-RYYRIK-ol NOP partial agonist hexapeptide 8.77±0.07 1.3
Ac-RYYRIK-NH2 8.22±0.07 5.1
Ac-RYYRIR-ol NOP hexapeptide analogues 8.14±0.03 6.1
Ac-RYYCitIK-ol 7.43±0.04 31
Ac-RYYCitIK-NH2 6.83±0.04 126
UFP-101 NOP antagonist peptide 7.71±0.04 16
J-113397 Non-peptide NOPr antagonist 6.61±0.05 206
Naloxone-benzoylhydrazone
Partial opioid agonist / NOPr antagonist
5.85±0.07 1196
Buprenorphine Opioid agonist-antagonist 5.34±0.11 3827
D-Ala3-Dynorphin(1-11) KOP selective opioid agonist peptide 5.12±0.11 6399
Table 6 Competition of [3H]Ac-RYYRIK-ol binding in rat brain membranes. *Calculated by Cheng-Prusoff equation
43
DISCUSSION
In this thesis detailed biochemical characterization of several PENK and PDYN
derived penta- and octapeptide enkephalin sequences have been described and their
selectivity for the opioid receptors has been evaluated. Other than opiod sequences, a
novel NOP receptor hexapeptide ligand Ac-RYYRIR-ol has been tested in both
biochemical and in-vitro pharmacological assays. Furthermore a full characterization
of our novel radiolabeled hexapeptide analogue [3H]Ac-RYYRIK-ol, selective for the
NOP receptor has been described by the use of neuronal and cultured epithelial cell
membrane preparations containing native or recombinant proteins.
Identification of the Novel Naturally Occurring Opioid Peptides
Interspecies variability of opioid peptides and their precursors point to the
significance of evolutionary changes and the need of comparative research. Altogether
39 PDYN sequences representing 36 animal species have so far been collected by
database searching. Non-allelic PDYN gene duplicates have been identified in the
tetraploid Xenopus laevis (Pattee et al., 2003). Organization and peptide sequence
variability appears more complex among PDYNs, since both the number and the
length of the opioid units are rather different within this precursor.
Isoleucine enkephalin is N-terminally located within the structure of xendorphin-1A.
This is the first opioid segment from the 5’ end of Xenopus PDYN-A cDNA sequence
encoding one of the two prodynorphin precursor polypeptides in this frog species
(Pattee et al., 2003). It is assumed, that Ile-enkephalin can be a naturally occurring
peptide in these animals, because an analogous ‘biosynthetic’ route producing Leu-
enkephalin by sequential enzymatic hydrolysis of larger PDYN fragments have been
reported in mammals (Traynor, 1987; Dixon and Traynor, 1993). Since the Ile-
enkephalin motifs are flanked by basic dipeptide repeats in each species listed in, the
formation of Ile-enkephalin may proceed through regular enzymatic cleavage. The
Xenopus laevis hexadecapeptide xendorphin-B1 produced naloxone reversible anti-
nociception measured by the acetic acid pain test in frogs (Stevens et al., 2009). In
addition, xendorphin-B1 displayed notable κ-opioid receptor affinity in radioligand
binding assays (Stevens et al., 2007). The heptapeptide YGGFMGY derived from the
zebrafish PENK has been shown to bind differentially to duplicate (ZFOR1 and
ZFOR4) DOP receptors from Danio rerio (Gonzalez-Nunez et al., 2007). Little is
44
known about the biological properties of the recently discovered atypical opioids.
Since there were no more data available on the bioactivity of the lately identified
endogenous opioids, we determined the binding and signaling properties of the two
novel enkephalins, i.e., the ’frog’ Ile-enkephalin and the ’lungfish’ Phe-enkephalin.
Such studies provide relatively quick and precise estimates on the basic characteristics
of the ligands without performing detailed pharmacological assays.
The unique structure of Phe-enkephalin has been detected in the brain cDNA
sequence encoding the African lungfish (Protopterus annectens) PDYN (Dores et al.,
2004). The Protopterus PDYN encodes for two copies of Leu-enkephalin and one-
one single copy of Met- and Phe-enkephalin. A full-length proenkephalin cDNA
(Protopterus PENK) was also cloned from the same species (Dores et al., 2000).
In our hand, these non-mammalian enkephalins showed slightly varying affinities in
different radioligand binding assays. All four enkephalins displayed high to moderate
affinities in the DOP receptor selective binding assays. As far as the general opioid
nature is concerned, Met-enkephalin seems the most potent, while Phe-enkephalin the
least efficacious peptide. Consequently, the KOP receptor preference of this peptide,
if any, needs further evaluations. The potency and the efficacy of Met-enkephalin
was by far the best in the [35S]GTPγS assays, but the overall differences among the
potencies of the peptides tested were smaller than in the competition binding
experiments.
In spite of the numerous reports on the correct chemical and immunohistological
identification of enkephalin-like peptides in invertebrates, there is no genomic or
cDNA sequence data describing the complete structure of any invertebrate enkephalin
precursor(s). However, enkephalin biosynthesis is well established in mammals
(Noda et al., 1982). Mammalian and other vertebrate enkephalins are regularly
produced by posttranslational enzymatic processing of their primary precursor
proenkephalin (PENK). Prodynorphin (PDYN) propeptides might be secondary
sources of enkephalins (Traynor, 1987; Bojnik et al., 2009a). Opioid peptides,
including enkephalins, also form families of closely related peptides, where several
members may occur in one animal species (Dores et al., 2002). This is due to gene or
exon duplications followed by mutations. Gene splicing and posttranslational
processing ultimately decides the gene product in a single cell of given species
(Holmgren and Jensen, 2001). The ancestral opioid receptor gene duplications
remarkably coincide with the origin of the four opioid peptide precursor genes
45
(Dreborg et al., 2008; Dores et al., 2002). Thus, the complete vertebrate opioid
system was already established in the first jawed vertebrates (Gnathostoma).
The organization of PENK is well conserved (Roberts et al., 2007), seven individual
enkephalin motifs exist in almost all PENK sequences of the 55 vertebrate species
studied so far. Our bioinformatical search with PENK has focused on the
phylogenetic diversity of the octapeptide enkephalin segment placed in the fourth
position (IVth) of the repetitive opioid units. The typical mammalian PENK
octapeptide is YGGFMRGL, but five other point-mutated orthologues have been
collected by analyzing mammalian PENK sequences. The mutations occurred within
the C-terminal tripeptide region of the octapeptides. No mutations on the enkephalin
pentapeptide unit was observed, so the opioid message region (Schwyzer, 1986)
carrying the structural basis of the biological activity is indeed the most conserved
part as suggested by Holmgren and Jensen (2001).
The amphibian Xenopus and Bombina octapeptides have the same structure as in the
platypus. However, the most abundant mammalian sequence YGGFMRGL is
apparently absent in the non-mammalian vertebrate taxa. Instead, data collected from
2 birds, 4 reptiles, 7 amphibians and 10 fish species reveal the occurrence of at least 7
such motifs those are not found in the mammalian PENKs. The octapeptide structure,
for instance, is YGGFMRSV in the chicken and in the Florida gar fish species. An
identical sequence is evidently present in one of the zebrafish (Danio rerio) PENKs,
although here the C-terminal cleavage site is missed (Gonzalez Nuñez et al., 2003).
Since the other, paralogous copy of the zebrafish PENK raised by gene or genom
duplication (Taylor et al., 2001) contains YGGFM sequence directly followed by the
RR endopeptidase recognition motif in the aligned position, no matured octapeptide
enkephalin is expected in this species. The PENK octapeptide motifs are flanked by
basic dipeptide repeats (KR or RR) in all other species, permitting the formation of
the mature oligopeptide by sequential enzymatic cleavage.
Four of the 14 different octapeptide enkephalins found so far in protein sequence
databases have been chemically synthesized and studied along with the human
sequence. Three novel structures are rather ubiquitous, as they are ranged in different
animals, while the fourth sequence of YGGFMNGF is solely present in a
cartilaginous fish species. From chemical aspects the Leu8→Tyr8 (frog) and
Leu8→Phe8 (shark) mutations are consistent with an aliphatic to aromatic change.
The Arg6→Asn6 replacement in the shark PENK is acconpanied by the loss of the
46
positively charged residue. The lungfish octapeptide YGGFMRSL contains a single
Gly7→Ser7 rather neutral mutation, while the YGGFMRSV peptide holds the
Gly7-Leu8→Ser7-Val8 duplicate point mutations.
The serine (Ser7) containing peptides displayed higher affinities in radioligand
binding assays, while the frog sequence (YGGFMRGY), also present in the platypus
PENK, seems to have lesser affinity. The opioid receptor-type selectivity pattern
supports that endogenous agonists are not by necessity the most specific ligands for a
particular receptor. The oxidation of the Met5 residue considerably reduced the
affinity of the enkephalins by shifting the competition curves to the right. Methionine
is a key amino acid that has numerous roles in essential vital processes. Moreover,
methionine oxidation might be biologically important during conditions of oxidative
stress (Bobrowski et al., 2008). Methionine oxidation in enkephalins (Turkall et al.,
1982) generally results in the decrease in biological response, although enhanced
antinociception of the oxidized enkephalin analogue [D-Ala2,Met5sulfoxide]enkephalin-
amide was also reported (Kiritsroy et al., 1983). The potency of Leu-enkephalin used
as control was the highest in the [35S]GTPγS assays, whereas Met-enkephalin and the
C-terminally extended octapeptides effectively stimulated the binding of [35S]GTPγS
to Gi/o proteins in rat brain membranes. Consistent with their full agonist effect, the
maximal stimulation values (% Emax, efficacy) of the octapeptides were around two
times higher over the basal activity.
As a conclusion for the newly recognized opioid sequences, the receptor binding and
G-protein activating properties of several novel, PDYN and PENK derived non-
mammalian opioid peptides were described, and compared to those of the well known
Met-enkephalin-Arg6-Gly7-Leu8 and the pentapeptide Met-enkephalin. Among the
pentapeptide enkephalin structures tested, Ile- and Phe-enkephalins were found to be
less effective than mammalian Met-enkephalin which exhibited the highest affinities
in receptor binding assays and produced the most efficacious G-protein stimulation in
brain membranes. Of the four octapeptide structures studied, the ‘chicken type’
peptide variant (YGGFMRSV) exhibited the highest affinities in receptor binding
assays. These novel endogenously occurring sequences represent further examples of
the natural diversity observed within the opioid peptide family. Importantly, these
new endogenous peptides represent further illustration of the chemical biodiversity
observed within the opioid peptide family. Research on the non-conventional
neuropeptide structures is essential from both phylogenetic and chemical-biochemical
47
viewpoints. One significance of these mutationally variable sequences is that they
represent a natural ‘combinatorial’ peptide library emerged by the evolution. The
various peptides evolved by gene mutations and natural selection offer template
sequences for structure-activity relationship studies. Inasmuch as the availability of
the genome sequencing data grows rapidly, an increasing impact of the phylogenetic
and bioinformatic studies on experimental biology is expected.
Pharmacological Characterization of the Ac-RYYRIR-ol
Intensive research has focused on the development of potent and selective NOP
receptor ligands, and resulted in various peptide and non-peptide agonist and
antagonist compounds (Zaveri et al., 2003; Lambert 2008). These discoveries have
been of great help for understanding the role of the N/OFQ system in a variety of
physiological processes, such as pain modulation, anxiety and depression, locomotor
activity, food intake, control of the cardiovascular and renal system, and so on
(Lambert, 2008, Jenk et al., 2000) .
In this thesis a novel NOP receptor targeting peptide analogue, Ac-RYYRIR-ol (Arg6
replacement in Ac-RYYRIK-ol; Kocsis et al., 2004) has been synthesized and tested
in both biochemical and pharmacological assays. The idea for replacing the
C-terminal lysine with an arginine unit was that the guanidinium group of Arg, in
addition to the electrostatic interaction, is capable of forming two supplementary
hydrogen bonds with an adjacent carboxylate group (Bajusz et al., 1980); therefore
higher binding affinities and receptor activation were expected. The significance of
the hexapeptides in the NOP receptor field is that they can elucidate the role of the N-
terminal phenylalanine in the N/OFQ heptadecapeptide and to investigate how
nociceptin and hexapeptide derivatives, which have completely different sequences,
can activate NOP receptor.
In receptor binding experiments done with [3H]Ac-RYYRIK-ol, the novel
hexapeptide Ac-RYYRIR-ol could effectively displace the radiolabelled analogue
with pKi value of 8.26, not very different from NOP receptor endogenous ligand
N/OFQ which was 8.69. Ac-RYYRIR-ol was not able to bind to MOP and DOP
receptors even up to high concentrations as demonstrated by the lack of displacement
of [3H]DAMGO and [3H]DIDI from rat brain membranes. Similar results were
obtained in the electrically stimulated tissues, where concentration response curves to
DPDPE in the mouse vas deferens (for DOP receptor selectivity) and to dermorphin in
48
the guinea pig ileum (for MOP receptor selectivity) were not affected in the presence
of Ac-RYYRIR-ol. In addition the high NOP selectivity of Ac-RYYRIR-ol is
suggested by the results obtained with UFP-101 that fully prevented the stimulatory
effects of Ac-RYYRIR-ol in GTP binding experiments performed in rat brain
membranes. Altogether these findings converge indicating that Ac-RYYRIR-ol
behaves as a high affinity and highly selective NOP receptor ligand. Ac-RYYRIR-ol
could stimulate the [35S]GTPγS binding using rat brain membranes. Efficacy of Ac-
RYYRIR-ol was much less compared to N/OFQ as it was previously described for
parent compound Ac-RYYRIK-ol under the same experimental conditions (Kocsis et
al., 2004). Stimulatory effect of both N/OFQ and Ac-RYYRIR-ol could be
antagonized with UFP-101, which is highly specific NOP receptor antagonist. This
result indicates that Ac-RYYRIR-ol stimulates selectively the G-proteins bound to the
NOP receptor. Ac-RYYRIR-ol similar to previously described NOP specific
hexapeptides produced both agonist and antagonist like effects in various assays used
in this study.
Mouse and rat vas deferens, and [35S]GTPγS binding studies also demonstrated the
NOP antagonist properties of Ac-RYYRIR-ol. In mouse vas deferens Ac-RYYRIR-
ol competitively antagonized the inhibitory effect and in the [35S]GTPγS binding the
stimulatory effect of N/OFQ. Similar results were reported using various
hexapeptides such as Ac-RYYRIK-NH2 and Ac-RYYRIK-ol (Berger et al., 1999,
Gündüz et al., 2006/2007). It has been also proven that this antagonistic effect is NOP
receptor specific as Ac-RYYRIK-NH2 was not able to inhibit the stimulatory effect
evoked by opioid receptor agonists (Berger et al., 1999). N/OFQ causes contractions
of the mouse proximal colon (Taniguchi et al. 1998) by inhibiting the tonic neuronal
release of nitric oxide (Menzies et al. 2000) and this preparation was used by many
authors for characterizing novel NOP related compounds (Rizzi et al., 1999, Menzies
et al., 1999, Gündüz et al 2006). In the mouse colon bioassay Ac-RYYRIR-ol
mimicked effect evoked by N/OFQ thus behaving like a very potent agonist in this
assay as it was previously described for the closely related hexapeptide Ac-RYYRIK-
ol (Gündüz et al., 2006/2007). Agonistic effect of Ac-RYYRIR-ol was also present in
the calcium mobilization experiments. Previously a characterization of the calcium
signaling via NOP receptor using several ligands including partial agonists was done
and shown to have overlapping potency as the Ac-RYYRIR-ol (Camarda et al.,
2009). Similar results have been shown for the Ac-RYYKWR-NH2 (Corbani et al.
49
2004). Ac-RYYRIR-ol and Ac-RYYRIK-ol was not able to induce NOP receptor
internalization. Partial agonists that can produce response without significant receptor
internalization might be good experimental tools in some cellular models of drug
tolerance.
Ac-RYYRIR-ol exhibited full (mouse colon) and partial (calcium mobilization,
[35S]GTPγS binding) agonist as well as antagonist (mouse and rat vas deferens)
properties at NOP receptors. These features of Ac-RYYRIR-ol are not surprising.
Similar behaviors have been reported for many other NOP receptor related peptides
such as; [Phe1ψ(CH2–NH)Gly2]-N/OFQ(1–13)–NH2, Ac-RYYRIK-NH2, and Ac-
RYYRIK-ol (Guerrini et al., 1998, Mason et al., 2001, Kocsis et al., 2004). One
explanation is the difference of NOP receptor densities in various preparations
(McDonald et al., 2003). Another possibility would be that low efficacy partial
agonist ligands might exert different degrees of activation depending on various
factors, such as the composition of the G-protein pool in the target tissue. It has been
demonstrated that partial agonists are able to produce low to full biological responses
(Berger et al., 2000).
Difference in the sensitivity of Ac-RYYRIR-ol in various assays, but not the N/OFQ,
might be explained by their likely diverse way of binding and activating the NOP
receptors as shown by photo-affinity labelling studies of the NOP receptor with radio-
iodinated probes (Bes and Meunier, 2003, Judd et al., 2004). Those findings have
indicated that the hexapeptides interacted with a region of Gln107-Gly-Thr-Asp-Ile-
Leu-Leu113 within the C-terminus of the second transmembrane domain (TM-II) in the
NOP receptor (Bes and Meunier, 2003, Guerrini et al., 1997), whereas N/OFQ
interacted with the region of Thr296-Ala-Val-Ala-Ile-Leu-Arg302, spanning the C-
terminus of EL-III and the N-terminus of transmembrane domain VII (Mouledous et
al., 2000). Recently, receptor-bound conformations of hexapeptide – NOP receptor
and N/OFQ – NOP receptor complexes have been calculated by computer aided
structural modeling (Akuzawa et al., 2007). This model describes how both sorts of
the peptides activate NOP receptor and supports that their docking conformations
have similar 3D structures. The receptor bound conformation of the N-terminal
FGGF message sequence in N/OFQ and the Ac-RYY fragment displayed fairly well
overlapping structural arrangements.
In conclusion, Ac-RYYRIR-ol displayed a complex biochemical and pharmacological
profile which is likely due to the low efficacy agonist nature of this unique
50
hexapeptide. It has been suggested that low partial agonism upon receptor – G-
protein coupling in native systems may be sufficient to evoke full biologic responses
(Berger et al., 2000). Exhibiting very good potencies and definite NOP receptor
selectivity in various assays makes Ac-RYYRIR-ol an attractive tool for
pharmacological and biochemical studies in the N/OFQ peptide receptor system.
Binding Characterization of the [3H]Ac-RYYRIK-ol
Moreover in this thesis in vitro characterization of a novel radiolabeled hexapeptide
analogue [3H]Ac-RYYRIK-ol, selective for the NOP receptor has been described.
Radioligands are essential tools in the G-protein coupled receptor (GPCR) research,
especially the 3H and 125I labelled ligands (Baker et al., 2000, Thomsen et al., 2000).
Binding characteristics and anatomical localization of the NOP receptors have been
studied by using a set of radioligands (reviewed by Dooley and Houghten, 2000).
Heterologous competition assays with various nociceptin analogues and some opioid
compounds revealed that [3H]Ac-RYYRIK-ol specific binding was effectively
inhibited by NOP receptor-selective ligands, whereas opioids displayed moderate to
negligible affinities. The highest affinities were obtained with the homologous ligand
Ac-RYYRIK-ol and N/OFQ, although the affinity of the Tyr1-replaced derivative
(Tyr1)N/OFQ was also very good. The iodinated analogue (I2Tyr1)N/OFQ, however,
displayed only modest affinity (Table 6) confirming the steric effect of the iodine
atoms discussed above. Among the opioids tested, naloxone-benzoylhydrazone was
the most efficient (Ki 1 µM), while the general opioid antagonist naloxone and the
MOP receptor agonist DAMGO were completely ineffective. The relative potency of
naloxone-benzoylhydrazone in competing reversibly the binding of [3H]Ac-RYYRIK-
ol is in line with the peculiar feature of this rather promiscous opiate (Cox et al.,
2005; Olianas et al., 2006; Connor and Kitchen, 2006). It should also be noted that
some non-peptide compounds such as buprenorphine and J-113397 displayed
considerably lower affinities than expected. This might be due to a potentially
different, or perhaps overlapping binding site(s) for N/OFQ and the hexapeptides
within the receptor as discussed below.
The results obtained in the present study revealed that the new tritiated peptide
analogue [3H]Ac-RYYRIK-ol recognized and labelled NOP receptors specifically in
rat brain membrane preparations and in cellular system expressing the recombinant
51
human NOP receptors. Importantly, this radioligand satisfied the criteria of
reversibility, saturability and relatively low non-specific binding necessary for
valuable radioprobes. These characteristics of [3H]Ac-RYYRIK, together with its
improved chemical and biological stability, and high specific radioactivity, make this
radioligand an inspiring tool for analyzing the properties and function of the NOP
receptor.
52
SUMMARY
Leu- and Met-enkephalin were the first endogenous opioid peptides identified in
different mammalian species including the human. Within the PENK structure two
Met-enkephalin sections are C-terminally elongated. One octapeptide sequence Tyr-
Gly-Gly-Phe-Met-Arg-Gly-Leu (YGGFMRGL) exists at the fourth position, while the
seventh, C-terminally located Met-enkephalin extension is present in heptapeptide
form. Comparative biochemical and bioinformatic evidence indicate that enkephalins
are not limited to mammals. Various PDYN and PENK sequences in lower
vertebrates revealed the presence of other endogenous opioid peptides in these
precursor polypeptides. Among the novel peptides Ile-enkephalin (Tyr-Gly-Gly-Phe-
Ile) was primarily observed in the African clawed frog (Xenopus laevis) PDYNs,
while the structure of Phe-enkephalin (Tyr-Gly-Gly-Phe-Phe) was predicted by
analyzing brain cDNA sequences encoding a prodynorphin of the African lungfish
(Protopterus annectens). Four of the orthologous Met-enkephalin octapeptide
sequences has been identified by which were: YGGFMRGY (three frog species,
platypus), YGGFMRSV (chicken, one fish species), YGGFMNGF (shark) and
YGGFMRSL (mouse, two lungfish species). All the novel sequences were chemically
synthesized and studied in receptor binding and G-protein activation assays performed
on rat brain membranes.
In various receptors binding assays performed on rat brain membrane preparations
both of the enkephalin peptides turned out to be moderate affinity opioids with a weak
preference for the DOP receptor sites. Phe-enkephalin of the lungfish displayed rather
unexpectedly low affinities toward the MOP and DOP, while exhibited moderate
affinity toward the KOP receptor. In receptor-mediated G-protein activation assays
measured by the stimulation of [35S]GTPγS binding, Met-enkephalin produced the
highest stimulation followed by Leu-enkephalin, Ile-enkephalin and Phe-enkephalin.
The overall binding and signalling profile of the novel octapeptides as well revealed
moderate opioid agonist activities and a rank order of potencies for the mu delta >>
kappa receptor binding sites. Peptides with the oxidized M(O) residue were found to
be less potent in both receptor binding and G-protein stimulation studies.
53
Nociceptin/orphanin FQ (N/OFQ) is an endogenous neuropeptide, which is widely
distributed in central and peripheral nervous system. Some N/OFQ sequence
unrelated hexapeptides can effectively bind to the NOP receptor and they were used
as template for structure activity studies that lead to discovery of the new NOP
selective ligands. In this thesis, the pharmacological profile of the novel hexapeptide
Ac-RYYRIR-ol was investigated using various in vitro assays including receptor
binding and G protein activation in rat brain membranes, mouse and rat vas deferens,
guinea pig ileum, mouse colon and Ca2+ mobilization assay in chinese hamster ovary
(CHO) cells co-expressing the human recombinant NOP receptor and the C-
terminally modified Gαqi5 protein.
Ac-RYYRIR-ol displaced [3H]Ac-RYYRIK-ol with high affinity and stimulated
[35S]GTPγS binding but showing lower maximal effects than N/OFQ. In antagonist
type experiments Ac-RYYRIR-ol inhibited the stimulatory effects induced by
N/OFQ. Meanwhile in mouse colon Ac-RYYRIR-ol produced concentration
dependent contractile effects with similar potency and maximal effects as N/OFQ and
finally, in the Ca2+ mobilization assay Ac-RYYRIR-ol displayed lower potency. We
have concluded that the novel NOP receptor selective hexapeptide Ac-RYYRIR-ol
has fine selectivity, high potency, furthermore agonist and antagonist effects toward
the NOP receptors which is likely due to its partial agonist pharmacological activity.
Radioreceptor binding studies were of [3H]Ac-RYYRIK-ol was conducted using
native neuronal NOP receptor preparation of rat brain membrane fractions and
recombinant human nociceptin receptor (hNOP) preparations from cultured CHO cells
stably expressing hNOP receptors. Results indicated that the new tritiated peptide
analogue [3H]Ac-RYYRIK-ol recognized and labeled NOP receptors specifically in
rat brain membrane preparations and in cellular system. This new radiprobe [3H]Ac-
RYYRIK, with high specific radioactivity, makes this radioligand an inspiring tool for
analyzing the properties and function of the NOP receptors.
54
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